Evolution and biogeography of gymnosperms

Full text

Review
Evolution and biogeography of gymnosperms
Xiao-Quan Wang
⇑
, Jin-Hua Ran
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
article info
Article history:
Received 30 June 2013
Revised 6 February 2014
Accepted 10 February 2014
Available online 22 February 2014
Keywords:
Phylogeny
Biogeography
Evolution
Gnetales
Conifers
Gymnosperms
abstract
Living gymnosperms comprise only a little more than 1000 species, but represent four of the five main
lineages of seed plants, including cycads, ginkgos, gnetophytes and conifers. This group has huge
ecological and economic value, and has drawn great interest from the scientific community. Here we
review recent advances in our understanding of gymnosperm evolution and biogeography, including
phylogenetic relationships at different taxonomic levels, patterns of species diversification, roles of
vicariance and dispersal in development of intercontinental disjunctions, modes of molecular evolution
in different genomes and lineages, and mechanisms underlying the formation of large nuclear genomes.
It is particularly interesting that increasing evidence supports a sister relationship between Gnetales
and Pinaceae (the Gnepine hypothesis) and the contribution of recent radiations to present species
diversity, and that expansion of retrotransposons is responsible for the large and complex nuclear
genome of gymnosperms. In addition, multiple coniferous genera such as Picea very likely originated
in North America and migrated into the Old World, further indicating that the center of diversity is
not necessarily the place of origin. The Bering Land Bridge acted as an important pathway for dispersal
of gymnosperms in the Northern Hemisphere. Moreover, the genome sequences of conifers provide an
unprecedented opportunity and an important platform for the evolutionary studies of gymnosperms,
and will also shed new light on evolution of many important gene families and biological pathways
in seed plants.
Ó2014 Elsevier Inc. All rights reserved.
Contents
1. Introduction .......................................................................................................... 25
2. Diversity and classification of gymnosperms . . . . . . . . . ....................................................................... 25
3. Phylogeny and evolution of gymnosperms. . . . . . . . . . . ....................................................................... 25
3.1. Origin and diversification . . . . . . . . . . . . . ............................................................................. 25
3.2. Phylogenetic reconstruction . . . . . . . . . . . ............................................................................. 28
3.2.1. The phylogenetic position of Gnetales . . . . . . .................................................................. 28
3.2.2. Phylogenetic studies of other gymnosperms . .................................................................. 30
3.3. Molecular and genome evolution. . . . . . . ............................................................................. 31
3.3.1. Chromosomal and genome size variation . . . . .................................................................. 31
3.3.2. Composition and evolutionary patterns of the three genomes of gymnosperms . . . . . . . . . . . ............................ 32
4. Biogeography of gymnosperms . .......................................................................................... 33
4.1. Disjunctive distribution in the two hemispheres . . . . . . . . . . ............................................................. 33
4.2. Disjunctive distribution in the Southern Hemisphere. . . . . . . ............................................................. 34
4.3. Disjunctive distribution in the Northern Hemisphere. . . . . . . ............................................................. 34
4.4. Biogeographic difference between the two hemispheres . . . . ............................................................. 35
5. Concluding remarks . . . . . . . . . . .......................................................................................... 35
Acknowledgments . . . . . . . . . . . .......................................................................................... 36
http://dx.doi.org/10.1016/j.ympev.2014.02.005
1055-7903/Ó2014 Elsevier Inc. All rights reserved.
⇑
Corresponding author. Address: State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan,
Beijing 100093, China. Fax: +86 10 62590843.
E-mail address: xiaoq_wang@ibcas.ac.cn (X.-Q. Wang).
Molecular Phylogenetics and Evolution 75 (2014) 24–40
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

Appendix A. Supplementary material .................................................................................... 36
References . . . . ....................................................................................................... 36
1. Introduction
Gymnosperms are of great ecological and economic importance,
although this ancient and widespread plant lineage currently com-
prises only a little more than 1000 species that are two to three or-
ders of magnitude lower than the approximately 300,000 species
of extant angiosperms. Also, from the evolutionary perspective,
studies of angiosperms depend a lot on our knowledge of gymno-
sperms given the sister relationship between the two groups. How-
ever, the evolutionary study of gymnosperms is still facing great
difficulties in the genomic era due to the large genome size, high
heterozygosity and long generation time of this group, although
a draft assembly of the Norway spruce (Picea abies) genome was
generated by Nystedt et al. (2013) and several comparative genom-
ics projects involving gymnosperms are being carried out, such as
the 1000 Plants (oneKP or 1KP) Initiative (http://www.onekp.com/
), the Plant Genomics Consortium (http://sciweb.nybg.org/sci-
ence2/GenomicsLab.asp), and the Conifer Genome Network
(http://www.pinegenome.org/index.php). Nevertheless, in recent
years, fascinating progress has been made in our understanding
of evolution and biogeography of gymnosperms, which inspires
us to write this review. For a better understanding of the content,
we first give a brief introduction of the diversity and classification
of gymnosperms. Then, we focus on: (1) Phylogeny and evolution
of gymnosperms, including evolutionary history, phylogenetic
relationships, and molecular and genome evolution; (2) Historical
biogeography of gymnosperms.
2. Diversity and classification of gymnosperms
Living gymnosperms are distributed in all continents except
Antarctica, of which two-thirds are conifers, a group that consti-
tutes over 39% of the world’s forests (Armenise et al., 2012). The
gymnosperms play major roles in global carbon cycles, provide
important sources of timber, resins and even drugs and foods (Zon-
neveld, 2012c; Murray, 2013), and are crucial to preventing soil
erosion. Additionally, they are a mainstay of gardening.
Gymnosperms represent four of the five main lineages of seed
plants, i.e., cycads, ginkgos, gnetophytes and conifers (including
cupressophytes and Pinaceae), and were recently classified into
four subclasses (Ginkgoidae, Cycadidae, Pinidae and Gnetidae) un-
der the class Equisetopsida (Chase and Reveal, 2009). They com-
prise 12 families, 83 genera (Christenhusz et al., 2011), and about
1000 species (Table 1), including ca 297–331 species of cycads in
10 genera, one extant ginkgophyte, 80–100 gnetophytes in three
genera, and ca 614 species of conifers in 69 genera (Farjón, 2010;
Christenhusz et al., 2011). Among these genera, 34 (40.96%) are
monotypic, 22 (26.5%) have only two to five species, and only three
(Cycas,Pinus and Podocarpus) harbor near or more than 100 species
(Table 1, and Fig. 1). It is interesting that half (45) of the genera oc-
cur in Asia and 31 in Australia (continent), and the vast majority of
the monotypic genera are found in these two continents (Fig. 2).
As the largest lineage of gymnosperms, conifers were divided
into seven families by Pilger (1926), including Taxaceae,
Podocarpaceae, Araucariaceae, Cephalotaxaceae, Pinaceae,
Taxodiaceae and Cupressaceae. However, Eckenwalder (1976)
proposed a merger of Taxodiaceae and Cupressaceae based on the
phenetic analysis and Hayata (1931) proposed to place Sciadopitys
in a separate family (Sciadopityaceae), and these views have been
adopted in most of the following classification schemes of
gymnosperms (e.g., Farjón, 2001, 2005; Christenhusz et al., 2011)
and supported by most non-molecular and molecular phylogenetic
studies (e.g., Hart, 1987; Price and Lowenstein, 1989; Brunsfeld
et al., 1994; Gadek et al., 2000; Yang et al., 2012). Currently, it is still
controversial whether Taxaceae and Cephalotaxaceae should be
merged into a single family (e.g., Quinn et al., 2002; Hao et al.,
2008; Christenhusz et al., 2011; Ghimire and Heo, 2014). Recently,
Eckenwalder (2009) and Farjón (2010) published two very valuable
books on all conifers, recognizing 546 and 615 species, respectively.
Although both books provided an identification guide to each spe-
cies, Farjón incorporated more recent advances in the systematics
of conifers and recognized more species, genera, and even families
than Eckenwalder. For example, Cephalotaxaceae and
Phyllocladaceae were recognized by Farjón (2010), but were put
into Taxaceae and Podocarpaceae, respectively, by Eckenwalder
(2009). In addition, Farjón (2010) recognized three extra genera,
Pilgerodendron and Xanthocyparis in Cupressaceae and Sundacarpus
in Podocarpaceae. At the family level, Farjón (1990, 2005) published
two excellent monographs on Pinaceae and Cupressaceae s.l.,
respectively. An interesting thing is that recent phenotypic and
molecular phylogenetic studies do not support the monophyly of
Cupressus (Cupressaceae). Adams et al. (2009) divided this genus
into two lineages, including Cupressus s.s. comprising the Old World
species and a new genus (Hesperocyparis) comprising the New
World species that are closely related to two small controversial
genera, i.e., Callitropsis from northwestern North America and
Xanthocyparis from northern Vietnam (Little, 2006; Yang et al.,
2012). However, except that Xanthocyparis was accepted by Farjón
(2010), the other three genera (Callitropsis,Hesperocyparis and
Xanthocyparis) were not accepted by Eckenwalder (2009), Farjón
(2010) and Christenhusz et al. (2011). At present, it is widely
accepted that conifers comprise two major clades, Pinaceae and
the remaining non-Pinaceae conifers (Conifer II or Cupressophytes)
(see Section 3.2, phylogenetic reconstruction), with Pinaceae and
Podocarpaceae representing the first and second largest families
(Farjón, 2001; Knopf et al., 2012).
The gnetophytes comprise three families (Ephedraceae,
Gnetaceae and Welwitschiaceae), each containing a single genus
(Table 1). Compared to gnetophytes and conifers, there were more
debates on the classification of cycads. Initially, all living species of
cycads were placed in a single family, the Cycadaceae (see reviews
by Stevenson, 1990 and Jones, 2002). However, afterwards, three to
four families, including Cycadaceae, Stangeriaceae, Zamiaceae and
Boweniaceae, were recognized by different authors (Johnson,
1959; Stevenson, 1981, 1990, 1992). The Boweniaceae was erected
by Stevenson (1981), but was mergered into Stangeriaceae by
Stevenson (1992). Recently, molecular phylogenetic studies
support a division of the 10 cycad genera into two families
(Cycadaceae and Zamiaceae) (Treutlein and Wink, 2002; Hill
et al., 2003; Chaw et al., 2005; Zgurski et al., 2008; Nagalingum
et al., 2011; Salas-Leiva et al., 2013), although the genus status of
Chigua is still accepted by some researches (http://plantnet.rbg-
syd.nsw.gov.au/PlantNet/cycad/) (see review by Osborne et al.,
2012).
3. Phylogeny and evolution of gymnosperms
3.1. Origin and diversification
Based on fossil evidence and molecular clock calibration, the
divergence between gymnosperms and angiosperms could be
dated to about 300–350 million years ago (Mya) in the
X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40 25

Table 1
Extant genera of gymnosperms and their biological and genome information.
Families Genera Number of species Genome size (pg/2C) No. of chromosomes (1n) Distribution
Farjón (2010)
e
Eckenwalder (2009)
e
Pinaceae
Abies 47 40 35.28 ± 2.38 12 AF (N), AS, EU, NA
Cathaya 1 1 49.5 12 AS
Cedrus 3 2 32.16 ± 0.65 12 AF (N), AS, EU
Keteleeria 3 2 48.4 12 AS
Larix 11 10 25.17 ± 3.64 12 AS, EU, NA
Nothotsuga 1 1 12 AS
Picea 38 29 36.20 ± 2.76 12 AS, EU, NA
Pinus 113 97 52.86 ± 8.21 12 AF (N), AS, EU, NA
Pseudolarix 1 1 52.2 22 AS
Pseudotsuga 4 4 38.1 12, 13 AS, NA
Tsuga 9 8 38.14 ± 3.46 12 AS, NA
Total 231 195
Cupressaceae
Actinostrobus 3 3 21.30 11 AU
Athrotaxis 3 2 20.17 ± 0.06 11 AU
Austrocedrus 1 1 21.80 11 SA
Callitris 15 17 18.43 ± 2.18 11 AU
Calocedrus 4 3 33.80 ± 3.21 11 AS, NA
Chamaecyparis 5 5 21.16 ± 2.88 11 AS, NA
Cryptomeria 1 1 22.48 ± 0.55 11 AS
Cunninghamia 2 2 26.92 ± 2.01 11 AS
Cupressus 15 17 23.64 ± 2.06 11 AF (N), AS, EU, NA
Diselma 1 1 18.1 11 AU
Fitzroya 1 1 35 22 SA
Fokienia 1 1 25.05 11 AS
Glyptostrobus 1 1 19.97 11 AS
Juniperus 53 54 27.33 ± 8.51 11, 22 AF, AS, EU, NA
Libocedrus 5 6 35.70 ± 6.60 11 AU
Metasequoia 1 1 22.08 11 AS
Microbiota 1 1 20.34 11 AS
Neocallitropsis 1 1 25.5 11 AU
Papuacedrus 1 1 24 11 AU
Pilgerodendron
a
1 0 11 SA
Platycladus 1 1 21.86 ± 1.34 11 AS
Sequoia 1 1 64.27 33 NA
Sequoiadendron 1 1 19.85 11 NA
Taiwania 1 1 30.22 ± 6.83 11 AS
Taxodium 2 2 19.00 ± 1.27 11 NA
Tetraclinis 1 1 25.7 11 AF (N), EU
Thuja 5 5 25.07 ± 1.20 11 AS, NA
Thujopsis 1 1 27.93 11 AS
Widdringtonia 4 4 21.35 ± 0.50 11 AF
Xanthocyparis
a,b
2 0 22.80 11 AS, NA
Total 135 136
Taxaceae
Amentotaxus 6 2 60.4 18 AS
Austrotaxus 11 _ AU
Pseudotaxus 1 1 34.6 12 AS
Taxus 10 8 23.06 ± 0.77 12 AS, EU, NA
Torreya 6 6 43.97 ± 0.71 11, 12 AS, NA
Total 24 18
Cephalotaxaceae
a
Cephalotaxus 8 5 50.7 12 AS
Sciadopityaceae
Sciadopitys 1 1 41.6 10 AS
Araucariaceae
Agathis 17 15 29.25 ± 3.32 13 AS, AU
Araucaria 19 19 33.33 ± 6.27 13 AU, SA
Wollemia 1 1 27.87 13 AU
Total 37 35
Podocarpaceae
Acmopyle 2 2 15.65 ± 2.62 10 AU
Afrocarpus 5 2 10.90 ± 0.99 12 AF
Dacrycarpus 9 9 23.45 ± 18.03 10 AS, AU
Dacrydium 22 21 17.40 ± 9.12 10 AS, AU
26 X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40

Carboniferous (Hedges et al., 2006; Won and Renner, 2006; Clarke
et al., 2011; Crisp and Cook, 2011; Magallón et al., 2013). The five
main lineages of gymnosperms (cycads, ginkgos, cupressophytes,
Pinaceae and gnetophytes) separated from each other during the
Late Carboniferous to the Late Triassic (311–212 Mya), which were
much earlier than the occurrence of the earliest extant angio-
sperms (Magallón et al., 2013). Although only about 1000 extant
species of gymnosperms inhabit our planet, gymnosperms were
dominant through much of the Mesozoic.
Considering the earliest fossil record of gymnosperms from the
Palaeozoic (reviewed in Won and Renner, 2006) and the highly
conserved morphology of this group, with a number of species
being referred to as ‘‘living fossils’’, such as Metasequoia glyptostro-
boides,Ginkgo biloba (Niklas, 1997) and cycads (Norstog and
Nicholls, 1997), scientists have long assumed that much of the spe-
cies diversity of living gymnosperms is relictual, representing the
last remnants of their prosperous past (Florin, 1963; Hill and
Brodribb, 1999; Miller, 1999; McLoughlin and Vajda, 2005; Keppel
et al., 2008; Xiao et al., 2010; Alvarez-Yepiz et al., 2011). However,
the gymnosperm diversity has experienced interesting pulses of
extinction and speciation (Davis and Schaefer, 2011). Most of
extant gymnosperm species, even genera, are much younger than
we thought before (Crisp and Cook, 2011; Nagalingum et al.,
2011). For example, cycads were often cited as an example of living
fossils, because it was believed that this lineage of gymnosperms
reached their greatest diversity during the Jurassic–Cretaceous
and then declined to the present diversity of around 300 species.
Unexpectedly, however, Nagalingum et al. (2011) found that living
Table 1 (continued)
Families Genera Number of species Genome size (pg/2C) No. of chromosomes (1n) Distribution
Farjón (2010)
e
Eckenwalder (2009)
e
Falcatifolium 6 5 22.40 10 AS, AU
Halocarpus 3 3 18.27 ± 4.45 9, 11, 12 AU
Lagarostrobos 1 1 30.40 15 AU
Lepidothamnus 3 3 12.00 ± 2.17 14, 15 AU, SA
Manoao 1 1 27.60 10 AU
Microcachrys 1 1 8.30 15 AU
Nageia 5 5 11.20 10, 13 AS, AU
Parasitaxus 1 1 18 AU
Pherosphaera/Microstrobos 2 2 8.50 ± 0.14 13 AU
Podocarpus 97 82 18.72 ± 2.58 10, 11, 17–19 AF, AS, AU, SA
Prumnopitys 9 8 14.13 ± 2.22 18, 19 AU, SA
Retrophyllum 5 4 11.80 10 AS, AU, SA
Saxegothaea 1 1 10.20 12 SA
Sundacarpus
a
1 0 _ AS, AU
Phyllocladaceae
a,b
Phyllocladus 4 5 20.18 ± 3.11 9 AS, AU
Total 178 156
Gnetaceae
Gnetum 39
f
6.73 ± 1.94 11, 22 AF, AS, SA
Welwitschiaceae
Welwitschia 1
f
14.40 21 AF
Ephedraceae
Ephedra 50
f
31.37 ± 5.91 7, 14, 28 AF, AS, EU,NA, SA
Ginkgoaceae
Ginkgo 1 1 23.50 12 AS
Cycadaceae
Cycas 107
c
103
d
26.83 ± 1.37 11 AF, AS, AU
Zamiaceae
Bowenia 2
c
2
d
33.48 ± 11.20 9 AU
Ceratozamia 27
c
18
d
63.25 ± 0.07 8 NA
Chigua
g
02
d
SA
Dioon 14
c
11
d
49.50 ± 1.13 9 NA
Encephalartos 65
c
64
d
58.36 ± 5.00 9 AF
Lepidozamia 2
c
2
d
57.80 ± 3.54 9 AU
Macrozamia 41
c
40
d
53.40 ± 0.57 9 AU
Microcycas 1
c
1
d
41.20 13 NA
Stangeria 1
c
1
d
29.64 8 AF
Zamia 71
c
53
d
35.76 ± 6.92 8–14 NA, SA
Total 224 194
AF, Africa; AF (N), North Africa; AS, Asia; AU, Australia (continent); NA, North America; SA, South America; EU, Europe.
Information of chromosome numbers mainly comes from Plant DNA C-values Database (http://data.kew.org/cvalues) and Murray (2013). The average genome size of each
genus was also calculated based on information from the Plant DNA C-values Database (before May 4, 2013).
a
Genus or family accepted by Farjón (2010) but not by Eckenwalder (2009).
b
Genus or family accepted by Farjón (2010) but not by Christenhusz et al. (2011).
c
Species number from Osborne et al. (2012).
d
Species number from the Cycad Page (CP) (http://plantnet.rbgsyd.nsw.gov.au/PlantNet/cycad/wlist.html).
e
Only numbers of conifer species were listed.
f
Species number from Price (1996).
g
Genus not accepted by Eckenwalder (2009) and Osborne et al. (2012) but by CP.
X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40 27

cycad species are not much older than about 12 million years, sug-
gesting recent synchronous radiation of a living fossil and coevolu-
tion between living cycads and their insect pollinators. In addition,
Crisp and Cook (2011) estimated the ages and diversification rates
of gymnosperm lineages, and found that living gymnosperm
groups are not ancient, occupy diverse habitats and some probably
survived after making adaptive shifts. Actually, the radiative speci-
ation in the middle to late Cenozoic with a very low interspecific
genetic differentiation has been reported in most of the studied
gymnospermous genera with multiple species, such as Agathis,
Araucaria,Cycas,Ephedra,Gnetum,Juniperus,Picea,Pinus, and Podo-
carpus (Hill, 1995; Wagstaff, 2004; Ran et al., 2006; Won and Ren-
ner, 2006; Willyard et al., 2007; Ickert-Bond et al., 2009; Biffin
et al., 2010; Mao et al., 2010; Crisp and Cook, 2011; Nagalingum
et al., 2011; Gaudeul et al., 2012; Leslie et al., 2012).
The low species diversity of extant gymnosperms could be
attributed to the Cenozoic extinctions, although recent radiations
have occurred in some lineages such as cycads (Crisp and Cook,
2011; Nagalingum et al., 2011). The extinctions at the Creta-
ceous-Palaeogene boundary affected major clades of gymnosperms
and angiosperms indifferently (Macphail et al., 1994; Niklas, 1997;
Wing, 2004; Crepet and Niklas, 2009). It was suggested that the
sharp cooling and drying of the global climate at the end of the Eo-
cene caused the extinction of several conifer and cycad lineages be-
cause gymnosperms probably occupied warmer and wetter
aseasonal environments during much of their early history (Hill,
1998, 2004; Hill and Brodribb, 1999). The climatic changes also
stimulated some lineages that survived to undergo successful
adaptive shifts and rediversify in new environments (Hill and Bro-
dribb, 1999; Pittermann et al., 2012), such as Callitris (Paull and
Hill, 2010) and Macrozamia (Carpenter, 1991). Leslie et al. (2012)
found that the lineages of gymnosperms that diversified mainly
in the Southern Hemisphere show a significantly older distribution
of divergence ages than their counterparts in the Northern Hemi-
sphere, which could be attributed to the fundamental differences
between the two hemispheres in the distribution of oceans and
landmasses. They also inferred that the complex patterns of migra-
tion and range shifts during climatic cycles in the Neogene could
have led to elevated rates of speciation and extinction and resulted
in the abundance of recent divergence in northern clades, whereas
the scattered persistence of mild, wetter habitats in the Southern
Hemisphere might have favored the survival of older lineages (Les-
lie et al., 2012).
In the long history of diversification, the morphology of gymno-
sperms has also undergone frequent parallel or convergent evolu-
tion, such as erect cones, deciduous needles and seed scale
abscission in Pinaceae (Wang et al., 2000), quadrangular leaves
linked to drought-tolerance in Picea (Ran et al., 2006), winged
seeds adapted to wind-dispersal in Pinus, small and imbricate
leaves that can minimize water loss in Cupressaceae s.l. (Pitter-
mann et al., 2012), colorful and fleshy cone bracts adapted to ani-
mal dispersal in Ephedra (Hollander and Vander Wall, 2009). In
particular, Lovisetto et al. (2012) found that a series of different
MADS-box genes are involved in the formation of gymnosperm
fruit-like structures, and the same gene types have been recruited
in the two phylogenetically distant species Ginkgo biloba and Taxus
baccata to make fleshy structures with different anatomical origins.
A further study showed that the gymnosperm B-sister genes may
have a main function in ovule/seed development and a subsidiary
role in the formation of fleshy fruit-like structures that have an
ovular origin, as in Ginkgo (Lovisetto et al., 2013). Therefore, these
non-synapomorphic characters should be used very carefully in
phylogenetic reconstruction and infrageneric classification.
3.2. Phylogenetic reconstruction
A number of molecular phylogenies of gymnosperms have been
published since the first molecular study that supports the sister
relationship between extant gymnosperms and angiosperms was
conducted by Hori et al. (1985) using 5S rRNA sequences. While
some interfamilial and intergeneric relationships have been re-
solved, more interesting phylogenetic hypotheses, especially on
the evolutionary relationships of deep branches of gymnosperms,
have been proposed and hotly debated, such as the phylogenetic
position of gnetophytes (Chaw et al., 1997, 2000; Bowe et al.,
2000; Mathews, 2009; Ran et al., 2010; Yang et al., 2012). In recent
years, low-copy nuclear genes and EST sequences have been used
in phylogenetic reconstruction of gymnosperms (Lee et al., 2011;
Yang et al., 2012; Xi et al., 2013), but most of previous studies still
used cytoplasmic DNA markers and/or nuclear ribosomal DNA
(nrDNA). The followings are the main progresses on phylogenetic
reconstruction.
3.2.1. The phylogenetic position of Gnetales
In the plant tree of life, Palmer et al. (2004) presented six major
unsolved problems, of which the most radical, shocking, and con-
troversial one was the placement of Gnetales, a small and morpho-
logically unique group of gymnosperms. After nine years, the
Fig. 1. Frequency distribution of species numbers of all gymnospermous genera.
The species numbers of coniferous genera follow Farjón (2010), while those of
cycad genera follow Osborne et al. (2012). The detailed information of each genus is
shown in Table 1.
Fig. 2. Number of gymnospermous genera in different continents and their species
richness. The distribution and species number of each genus correspond to Table 1.
N, number of species in a genus.
28 X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40

phylogenetic position of Gnetales still remains enigmatic, although
many efforts have been made to resolve this problem.
The Gnetales includes the three isolated genera Ephedra,Gne-
tum, and Welwitschia. Based on the early cladistic analyses using
morphological characters, Gnetales was considered to be the sister
of angiosperms (the anthophyte hypothesis, Fig. 3A) (Doyle and
Donoghue, 1986; Rothwell and Serber, 1994; reviewed by Doyle,
1998). The anthophyte hypothesis seemed to be supported by
some morphological, anatomical and reproductive characters, such
as net-veined leaves, vessels in the wood, double fertilization and
the simple, unisexual, flower-like structures, but later studies re-
vealed that these character similarities between Gnetales and
angiosperms are not really homologous or due to parallel evolution
(Winter et al., 1999; Donoghue and Doyle, 2000; Mundry and Stut-
zel, 2004). Moreover, this hypothesis has been questioned by most
molecular studies (Supplementary material S1) (e.g., Magallón and
Sanderson, 2002; Soltis et al., 2002; Burleigh and Mathews, 2004,
2007a; Ran et al., 2010; Zhong et al., 2010; Lee et al., 2011; Wu
et al., 2011; Xi et al., 2013). Although angiosperms and Gnetales
were grouped together in several studies using ribosomal DNA or
the third codon of the PHY gene, the results were very weakly sup-
ported statistically (Stefanoviac et al., 1998; Rydin et al., 2002;
Schmidt and Schneider-Poetsch, 2002), and could be caused by
some limitations of ribosomal DNA such as many paralogous cop-
ies (Zhang et al., 2012) or the substitutional saturation in PHY (Xia
et al., 2003). Nevertheless, it remains interesting that the antho-
phyte hypothesis is supported by the slowly-evolving ribosomal
DNA. In particular, Burleigh and Mathews (2007b) found bias
against recovering the anthophyte hypothesis in the molecular
data.
Some molecular studies supported Gnetales as sister to the
other seed plants (Supplementary material S1), especially when
some chloroplast DNA (cpDNA) sequences (all or the third codon
positions) were used and the maximum parsimony (MP) method
was used in phylogenetic reconstruction (e.g., Sanderson et al.,
2000; Rydin et al., 2002; Soltis et al., 2002; Rai et al., 2003,
2008). This topology was known as the Gnetales-sister hypothesis
by Burleigh and Mathews (2004) or the gnetales-sister II hypothe-
sis by Braukmann et al. (2009). For clarity, we suggest that it be
called ‘‘the gnetales-other seed plants hypothesis’’ (Fig. 3B). More-
over, some studies indicated Gnetales as sister to the rest of gym-
nosperms by analyzing cytoplasmic or nuclear genes (Hasebe et al.,
1992; Frohlich and Estabrook, 2000; Schmidt and Schneider-Poet-
sch, 2002; Burleigh and Mathews, 2004; also see Supplementary
material S1). It was referred as the Gnetales-sister I hypothesis
(Braukmann et al., 2009), and is hereafter called ‘‘the gnetales-
other gymnosperms hypothesis’’ (Fig. 3C). This hypothesis was also
strongly supported by a couple of recent phylogenomic studies
that used combined sequences mostly from expressed sequenced
tags (ESTs) (Cibrián-Jaramillo et al., 2010; Lee et al., 2011). Cur-
rently, neither the gnetales-other seed plants hypothesis nor the
gnetales-other gymnosperms hypothesis is widely accepted. One
of the most important reasons is that the MP method is more easily
affected by long branch attraction (LBA) than maximum likelihood
(ML) and Bayesian inference (BI) (Felsenstein, 1978; Hendy and
Penny, 1989; Huelsenbeck, 1995). Another reason is that the data-
sets used in the phylogenomic analyses contained too many miss-
ing data, which might perturb phylogenetic inference (Roure et al.,
2013). However, while recognizing the shortcomings in data anal-
ysis, we should keep in mind that these factors are also problems
Fig. 3. Five main hypotheses of the phylogenetic position of Gnetales. (A) the anthophyte hypothesis; (B) the gnetales-other seed plants hypothesis; (C) the gnetales-other
gymnosperms hypothesis; (D) the Gnetifer hypothesis; and (E) the Gnepine hypothesis.
X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40 29

underlying the phylogenetic inference of seed plants, a group
experiencing a long evolutionary history and many extinction
events.
Most molecular phylogenetic studies indicate that Gnetales has
a close relationship with conifers. Some of them support the Gne-
tifer hypothesis, i.e., Gnetales sister to conifers (Fig. 3D), based
mainly on analyses of nrDNA (Chaw et al., 1997; Soltis et al.,
1999) and the mitochondrial rps3 gene (Ran et al., 2010). However,
the Gnepine hypothesis, i.e., Gnetales sister to Pinaceae (Fig. 3E,
Supplementary material S1), is supported by many more studies
after eliminating bias in data analyses (Bowe et al., 2000; Chaw
et al., 2000; Gugerli et al., 2001; Magallón and Sanderson, 2002;
Burleigh and Mathews, 2004; Hajibabaei et al., 2006; Zhong
et al., 2010; Wu et al., 2011; Burleigh et al., 2012; Xi et al.,
2013), although some results are locus-dependent. Moreover, the
Gnepine hypothesis is supported by the loss of all ndh genes,
rpl16 and two introns of clpP in the chloroplast genomes shared be-
tween Gnetales and Pinaceae (Tatsuya et al., 1994; Braukmann
et al., 2009; Wu et al., 2009). Interestingly, Zhong et al. (2010) ob-
tained a tree topology supporting a sister relationship between
Gnetales and cupressophytes (the Gnecup hypothesis) when all
chloroplast genes were used (Supplementary material S1). How-
ever, when they discarded some fast-evolving genes and three
genes with many parallel amino acid substitutions between Gne-
tales and cupressophytes, the topology changed to support the
Gnepine hypothesis. Although the nearly complete loss of one of
the inverted repeats (IRs) in all conifer chloroplast genomes
seemed to support the monophyly of conifers (Raubeson and Jan-
sen, 1992; Tsudzuki et al., 1992; Hirao et al., 2008; McCoy et al.,
2008), this structural mutation occurred in different IRs of Pinaceae
and Cupressophytes, and thus could not be used as a homologous
character or a synapomorphy (Wu et al., 2011; Wu and Chaw,
2013).
Recent advances in genome sequencing technologies, especially
next generation sequencing, have facilitated rapid sequencing of a
complete genome, transcriptome or large number of cDNA se-
quences from non-model organisms, providing a massive amount
of information for phylogenetic analyses. Phylogenomic analyses
have been widely used to reconstruct the tree of life, including to
resolve the phylogenetic position of Gnetales (de la Torre-Barcena
et al., 2009; Cibrián-Jaramillo et al., 2010; Zhong et al., 2010; Lee
et al., 2011; Wu et al., 2011, 2013; Burleigh et al., 2012). It is inter-
esting that most studies based on concatenated protein-coding nu-
clear genes (ESTs) support the gnetales-other gymnosperms
hypothesis (de la Torre-Barcena et al., 2009;Cibrián-Jaramillo
et al., 2010;Lee et al., 2011) whereas the studies based on chloro-
plast genes support the Gnepine hypothesis after data treatment
(Zhong et al., 2010; Wu et al., 2011). This incongruence could have
two explanations. First, for gymnosperms, the nuclear genome se-
quence is currently unavailable, and the available EST sequences
cannot well represent the nuclear genome except in some species
of Pinaceae (Mackay et al., 2012). Therefore, it is difficult to obtain
enough real orthologous nuclear genes from all main lineages of
gymnosperms for phylogenetic reconstruction. For example, Lee
et al. (2011) analyzed 12,469,970 amino acid sites from 150 species
across land plants. However, according to our reanalysis of the
data, none of the sites was left after removing the poorly aligned
positions in the alignment using Gblocks (Talavera and Castresana,
2007). Moreover, only 3688 and 1864 sites can be well aligned be-
tween Gnetophytes and angiosperms, and between Gnetophytes
and Pinaceae, respectively, and in particular the two alignments
have only about 130 overlapped sites (our unpublished data). Sec-
ond, the incongruence might have resulted from systematic errors
(Jeffroy et al., 2006), although phylogenomic analyses could some-
times improve the resolution of trees and solve difficult phyloge-
netic questions.
Systematic errors cannot be removed by increasing data, and,
on the contrary, may grow with increased size of datasets (Rodrí-
guez-Ezpeleta et al., 2005), owing to sequence composition biases
among lineages and sequence heterotachy (Wu et al., 2011). Bur-
leigh and Mathews (2004) found that both MP and ML trees sup-
ported the Gnepine hypothesis when fast-evolving positions
were removed from a 13-locus concatenated seed plant dataset,
and Zhong et al. (2010) obtained a sister relationship between
Gntales and Pinaceae after excluding fast-evolving genes and par-
allel sites. In addition, Wu et al. (2011) got congruent and robust
tree topologies supporting the gentophytes-Pinaceae clade as sis-
ter of cupressophytes when the removal of highly heterotachous
genes alleviated the artifact of LBA. Moreover, different rootings
will directly influence the topology of phylogenetic trees, espe-
cially for the inference of relationships among major lineages of
seed plants. For instance, Donoghue and Doyle (2000) investigated
the effect of alternative rootings on inferred relationships of Gne-
tales, angiosperms and conifers, and dsicussed the anthophyte
hypothesis. Mathews et al. (2010) explored this issue using a dupli-
cate gene rooting in analyses of phytochrome amino acids of seed
plants, and yielded trees that unite cycads and angiosperms in a
clade.
3.2.2. Phylogenetic studies of other gymnosperms
Besides the great effort in resolving the phylogenetic position of
Gnetales, many phylogenetic studies have been conducted on
other gymnosperms. For instance, the monotypic genus Ginkgo is
the sole survivor of ginkgos that originated at least 270 Mya, and
its systematic position has been controversial for a long time (see
review by Wu et al., 2013). It was placed in the coniferophyte clade
sensu Chamberlain (1935), comprising conifers, cordaites, ginkgo-
phytes, and gnetophytes. Some studies suggested that Ginkgo is
closer to conifers than cycads based on comparative development
of the spermatozoids (Norstog et al., 2004) or intermediate be-
tween these two lineages based on embryogenesis (Wang et al.,
2011). However, most molecular phylogenetic studies based on
single or a few genes support Ginkgo as sister to a clade comprising
conifers and gnetophytes (e.g., Chaw et al., 2000; Hajibabaei et al.,
2006; Mathews, 2009; Ran et al., 2010). It is particularly interest-
ing that most recent phylogenomic analyses support a sister rela-
tionships between Ginkgo and cycads (e.g., Cibrián-Jaramillo
et al., 2010; Finet et al., 2010; Wu et al., 2013; Xi et al., 2013),
which is consistent with the morphological characters shared be-
tween the two groups, such as an haustorial pollen tube (Friedman,
1993) and multiflagellated sperms (Ikeno and Hirase, 1897; Bren-
ner et al., 2003).
For cycads, molecular phylogenies were previously constructed
using two chloroplast genes and nrDNA (Hill et al., 2003), 17 chlo-
roplast genes and associated noncoding regions (Rai et al., 2003;
Zgurski et al., 2008), matK+ITS/5.8S rDNA (Chaw et al., 2005),
matK+26S rDNA (Crisp and Cook, 2011), and PHYP (Nagalingum
et al., 2011), respectively. All of the phylogenies support the basal
position of Cycas and the division of cycads into two families,
which have been recognized in the new book of Osborne et al.
(2012). That is, Cycadaceae comprises the single genus Cycas, while
Zamiaceae includes the rest of nine genera. However, some inter-
generic relationships within the family Zamiaceae, especially the
phylogenetic positions of Bowenia,Dioon and Stangeria, still remain
unresolved, although the genus Dioon was placed in a basal posi-
tion in most phylogenies (Hill et al., 2003; Rai et al., 2003; Chaw
et al., 2005; Zgurski et al., 2008; Crisp and Cook, 2011). Very re-
cently, a relatively solid phylogeny of cycads was reconstructed
using five single-copy nuclear genes, in which, for Zamiaceae,
Dioon diverged first, followed by Bowenia, and then an encephalar-
toid clade (Macrozamia–Lepidozamia–Encephalartos) sister to a
zamioid clade (Salas-Leiva et al., 2013).
30 X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40

Pinaceae is the largest and economically most important family
in conifers (Farjón, 1990). The first molecular phylogeny of all its
11 genera was constructed by Wang et al. (2000), using paternal
chloroplast, maternal mitochondrial and biparental low-copy nu-
clear genes. They found that different gene trees are largely identi-
cal in topology except the phylogenetic position of Cedrus and an
unresolved trichotomy formed by Cathaya,Picea and Pinus, and
the three-genome phylogeny supports the division of the pine fam-
ily into two major groups Abies–Keteleeria–Nothotsuga–Tsuga–
Pseudolarix–(Cedrus) and Cathaya–Picea–Pinus–Pseudotsuga–Larix
that correspond well with the distribution (number and position)
of resin canals in the central vascular cylinder of the young taproot.
Gernandt et al. (2008) investigated the phylogenetic history of Pin-
aceae using two chloroplast genes (matK and rbcL) and non-molec-
ular characters, and also found that the position of Cedrus is
inconsistent among different analyses. Recently, based on compar-
ative chloroplast genomics, Lin et al. (2010) concluded that Cedrus
was sister to the Abies–Keteleeria clade, and Cathaya was closer to
Pinus than to Picea. However, it is interesting that Cathaya looks
like a hybrid between Picea and Pinus in sequences of many genes
(our unpublished data). Actually, Cathaya is also morphologically
intermediate between the two genera. Like Picea, the needles of
Cathaya do not form bundles and are spirally arranged. In particu-
lar, some species of Picea also have Cathaya-like flattened leaves
that may represent a symplesiomorphic character. On the other
hand, Cathaya is similar to Pinus in having axillary seed cones
and in reproductive characteristics (reviewed in Wang et al.,
1998). Therefore, the relationships among the three genera still
need more investigations.
For Conifer II, the interfamilial relationships were consistently
revealed by previous molecular phylogenetic studies. That is, the
two families Araucariaceae and Podocarpaceae diverged first, fol-
lowed by Sciadopityaceae, and then Taxaceae-Cephalotaxaceae
that is sister to Cupressaceae s.l. comprising Taxodiaceae and
Cupressaceae s.s.(Chaw et al., 1997, 2000; Bowe et al., 2000; Rai
et al., 2008; Ran et al., 2010; Crisp and Cook, 2011; Burleigh
et al., 2012; Yang et al., 2012). Yang et al. (2012) reconstructed
the phylogeny of gymnosperms using two sister single-copy nucle-
ar genes LFY and NLY that originated from a gene duplication in the
common ancestor of seed plants, and further used the two genes,
together with chloroplast matK and mitochondrial rps3 genes to
reconstruct the phylogeny of Cupressaceae s.l. represented by all
its 32 genera. The different gene trees generated are topologically
highly congruent, supporting the basal position of Cunninghamia
and the division of Cupressaceae s.l. into six or seven subfamilies,
as recognized in Gadek et al. (2000) and Farjón (2005), respec-
tively. However, there are still some topological conflicts among
different trees that need to be resolved, such as the positions of
Papuacedrus and Tetraclinis, and the relationship between Fokie-
nia–Chamaecyparis and Thuja–Thujopsis. The conflicts could be
attributed to insufficient resolution of the molecular markers or
historical hybridization. For example, the inconsistent relation-
ships among the three genera Sequoia,Sequoiadendron and Metase-
quoia revealed in different gene trees suggest an allopolyploid
origin for the hexaploid Sequoia by hybridization between Metase-
quoia and Sequoiadendron or an extinct taxodiaceous plant. This
inference is also supported by the reticulation among the three
genera shown in the network analysis of the LFY and NLY genes
(Yang et al., 2012). The published molecular phylogenies of other
families of Conifer II include Podocarpaceae (Kelch, 1998; Conran
et al., 2000; Knopf et al., 2012), Araucariaceae (Setoguchi et al.,
1998; Liu et al., 2009), and Taxaceae (Cheng et al., 2000; Wang
and Shu, 2000; Hao et al., 2008). All molecular studies support
Phyllocladus as a genus of Podocarpaceae rather than as an inde-
pendent family (Phyllocladaceae), and some intergeneric relation-
ships within Podocarpaceae, the second largest family of conifers
comprising 19 genera, have not been well resolved (Kelch, 1998;
Conran et al., 2000; Knopf et al., 2012).
It is also exciting that phylogenies of some genera of gymno-
sperms have been reconstructed based on extensive species sam-
pling and multiple gene markers, such as Picea (Ran et al., 2006),
Pinus (Gernandt et al., 2005; Parks et al., 2009, 2012), Cedrus (Qiao
et al., 2007), Larix (Wei and Wang, 2004), Tsuga (Havill et al., 2008),
Pseudotsuga (Wei et al., 2010), Juniperus (Mao et al., 2010), Thuja
(Peng and Wang, 2008), Ephedra (Rydin and Korall, 2009) and
Podocarpus (Knopf et al., 2012). For instance, Wei et al. (2010) un-
veiled the interspecific relationships within the genus Pseudotsuga
and found that the Taiwanese species P. wilsoniana might have
originated by hybridization between two lineages from mainland
China, using five cpDNA and two mtDNA fragments as well as
the nuclear gene LFY. However, surprisingly, most species of the
large genera such as Picea,Pinus,Podocarpus and Cycas originated
from recent radiation. For example, Ran et al. (2006) investigated
the evolutionary history of 33 species of Picea using two chloro-
plast and one mitochondrial genes, and fossil evidence. They found
that this genus experienced at least two radiative speciation events
that occurred in northeastern Asia and southwestern China,
respectively. Therefore, it is still very tough to resolve evolutionary
relationships of closely related species of gymnosperms due to
radiative speciation and frequent interspecific hybridization.
3.3. Molecular and genome evolution
3.3.1. Chromosomal and genome size variation
Gymnosperms are probably the best studied group of land
plants by far with regard to chromosome number and genome size
(Fig. 4). Among the 83 genera of gymnosperms, the genome size
has been estimated for 344 species representing all genera except
Nothotsuga,Parasitaxus,Pilgerodendron and Sundacarpus, while the
chromosome number has been reported from a number of species
covering all genera except Austrotaxus and Sundacarpus (Zonne-
veld, 2012a,b; Leitch and Leitch, 2013; Murray, 2013). Considering
that several reviews have discussed the genome size variation (e.g.,
Morgante and De Pauli, 2011; Leitch and Leitch, 2013), or the
karyotype variation and evolution in gymnosperms (e.g., Nkongolo
Fig. 4. Variation of genome size in 13 gymnosperm families. Cyc, Cycadaceae; Zam,
Zamiaceae; Gin, Ginkgoaceae; Gne, Gnetaceae; Eph, Ephedraceae; Wel, Welwit-
schiaceae; Pin, Pinaceae; Ara, Araucariaceae; Pod, Podocarpaceae; Sci, Sciadopity-
aceae; Cep, Cephalotaxaceae; Tax, Taxaceae; Cup, Cupressaceae. The data were
calculated based on information from the Plant DNA C-values Database (http://
data.kew.org/cvalues) before May 4, 2013.
X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40 31

and Mehes-Smith, 2012; Murray, 2013), here we only summarize
the main characteristics of gymnosperm genomes as follows.
One remarkable feature is the narrow variation range of chro-
mosome numbers, from 2n= 14 to 66 in wild plants. In Pinaceae,
all studied species except Pseudolarix amabilis (2n= 44) and Pseud-
otsuga menziesii (2n= 26) have the same chromosome number of
2n=2x= 24. In Cupressaceae s.l., all 30 genera have the same basic
chromosome number of x= 11, and all studied species show
2n=2x= 22 except 2n=6x=66 in Sequoia semperviens and
2n=4x=44 in Juniperus chinensis ‘Pfitzeriana’ and Fitzroya
cupressoides. The basic chromosome number shows the widest var-
iation in Podocarpaceae, but only ranges from x=9 to x=19 (Ta-
ble 1). The second remarkable feature is that all congeneric
species have the same basic chromosome number except Halocar-
pus,Lepidothamnus,Nageia,Podocarpus and Prumnopitys in Podo-
carpaceae, Pseudotsuga in Pinaceae, Torreya in Taxaceae, and
Zamia in Zamiaceae. Moreover, intraspecific variation of basic
chromosome number was very rarely reported, such as in Zamia
loddigesii (Vovides and Olivares, 1996). According to the study of
Zhou et al. (2000),x= 7 and x= 20 reported in Amentotaxus argo-
taenia (Chuang and Hu, 1963; Guan et al., 1993) could be wrong
counts. The third feature is the remarkable karyotype conservation
across species and genera (e.g., Hizume et al., 2002;Shibata and
Hizume 2008;Nkongolo and Mehes-Smith, 2012). For the fertile
interspecific hybrids in Larix,Pinus,Dacrydium and Podocarpus,no
chromosomal translocations were observed (Sax, 1932, 1960; Say-
lor and Smith, 1966; Quinn and Rattenbury, 1972; Wardle, 1972;
Williams et al., 2002; Eckenwalder, 2009). Comparative genetic
mapping also revealed a remarkable interspecific and intergeneric
conservation of gene distribution and order (Krutovsky et al., 2004;
Pelgas et al., 2006). The fourth feature is that chromosomes are ex-
tremely large. For example, the length of metaphase chromosomes
ranges from 6.4 to16.2
l
minPinus, 5.4 to14.5
l
minPicea, and 4.4
to 11.6
l
minLarix (Hizume, 1988). The fifth feature is that gen-
omes of gymnosperms are, on average, larger than those of other
land plant groups (Murray, 1998; Leitch et al., 2001; Ahuja,
2005; Ahuja and Neale, 2005). The mean genome size of gymno-
sperms is 1C = 18.08 pg, a value much larger than that of angio-
sperms (1C = 5.9 pg) (Leitch and Leitch, 2013). The smallest and
largest genomes were found in Gnetum (1C = 2.25 pg) and Pinus
(1C = 36 pg), respectively (Table 1). The sixth feature is that poly-
ploidy is exceedingly rare in gymnosperms (Ahuja, 2005; Williams,
2009; Fawcett et al., 2013; Murray, 2013), with a frequency of
about 4–5% (Khoshoo, 1959; Delevoryas, 1980; Wood et al.,
2009). There are only three natural polyploids in conifers, includ-
ing the hexaploid Sequoia semperviens and the two tetraploids Juni-
perus chinensis ‘Pfitzeriana’ and Fitzroya cupressoides (Ahuja, 2005),
although recent studies indicate that polyploidy seems to have
played a more important role than earlier envisioned in the speci-
ation of conifers (Zonneveld, 2012a). The rest of natural polyploids
are all from Ephedra, accounting for about 50–65% of the studied
species of the genus (Khoshoo, 1959; Huang et al., 2005).
3.3.2. Composition and evolutionary patterns of the three genomes of
gymnosperms
3.3.2.1. Nuclear genome. Aided by the development of sequencing
technologies, fully sequenced plant genomes are rapidly growing
in number. However, none of them is from gymnosperms owing
to the extremely large genome sizes in this group. For instance,
conifers have a genome size of 8–70 gigabases (Gb) (2C), which
is among the largest of any nonpolyploid plant species (http://da-
ta.kew.org/cvalues/). This, together with large amounts of repeti-
tive DNA, has limited efforts to produce a conifer reference
genome (Lorenz et al., 2012; Ritland, 2012), and early studies
strongly suggest that the genome of conifers is very different from
that of angiosperms in composition and structure (see review by
Mackay et al., 2012). Fortunately, a draft assembly of the Norway
spruce (Picea abies) genome, approximately 19.6 Gb, has been gen-
erated very recently based on a hierarchical strategy combining
fosmid pools with both haploid and diploid whole genome shotgun
(WGS) data, and RNA sequencing (RNA-Seq) data, and it is surpris-
ing that this large genome harbors only about 28,354 genes (Nys-
tedt et al., 2013). At the same time, Birol et al. (2013) reported an
assembling of the 20.8 Gb white spruce (Picea glauca) genome
based on the WGS data. Moreover, the genome sequence of the
loblolly pine (Pinus taeda, around 22 Gb (1C)), is expected to be re-
ported soon (Sederoff, 2013). The available data indicate that dif-
ferent conifers (Norway spruce, white spruce, Sitka spruce, and
loblolly pine) have very similar numbers of unigenes, less than
30,000 as in many angiosperms, including Arabidopsis thaliana (Rit-
land, 2012). This finding clearly does not support the previous
hypotheses that there are more than 225,000 genes in the genome
of Pinaceae (Kinlaw and Neale, 1997; Rabinowicz et al., 2005). The
overestimation by early researchers could be caused by including
degenerate retroelements (Morgante and De Pauli, 2011), and large
numbers of pseudogenes (Rabinowicz et al., 2005; Garcia-Gil,
2008; Kovach et al., 2010;Rigault et al., 2011; Nystedt et al., 2013).
Why do gymnosperms have a so large genome? It is well known
that polyploidy or chromosome duplication is an important mech-
anism for generating large genomes in angiosperms. However, the
gymnosperm genomes show no evidence of recent whole-genome
or chromosome duplication (Ohri and Khoshoo, 1986; Kovach
et al., 2010; Nystedt et al., 2013), and few polyploid species are
found in the gymnosperm lineages except Ephedra (Khoshoo,
1959, 1961; Ahuja, 2005). Moreover, the basic chromosome num-
ber variation in diploid gymnosperms is very likely resulted from
chromosome fission or fusion, such as in Douglas fir (Khoshoo,
1961; Fuchs et al., 1995; Pelgas et al., 2006; Pavy et al., 2012).
Although a whole-genome duplication was inferred to have oc-
curred in the common ancestor of seed plants around 350 Mya
(Jiao et al., 2011; Nystedt et al., 2013), it should not have only con-
tributed to the increase of genome size in gymnosperms.
The large and complex genome of gymnosperms could have
developed by expansion of retrotransposons (Morse et al., 2009;
Nystedt et al., 2013), which can increase the genome size in very
short time by dispersing to new locations in a copy-and-paste fash-
ion through an RNA intermediate. Based on reassociation kinetics,
Rake et al. (1980) found that 75% of a conifer genome could be
repetitive elements, and suggested that dramatic amplification of
noncoding DNA might have contributed to the origin of large gen-
ome sizes in gymnosperms. Recently, the large amount of repeti-
tive elements, especially the LTR (long terminal repeat)
retrotransposons such as Ty1-copia, Ty3-gypsy and Gymny, have
been recognized as the primary factor causing inflation of gymno-
sperm genomes (Friesen et al., 2001; Morse et al., 2009; Plomion
et al., 2011; Nystedt et al., 2013). The gypsy and copia elements
in Pinaceae are distributed at the chromosome ends or are associ-
ated with 18S rDNA and centromeric regions (Friesen et al., 2001;
Morse et al., 2009; Grover and Wendel, 2010; Plomion et al., 2011),
which is fundamentally different from angiosperms (Leitch and
Leitch, 2013). It is particularly interesting that the transposable
element diversity is shared among extant conifers. In addition, it
has been confirmed that 24-nucleotide short RNAs (sRNAs), a class
of sRNA that can silence transposable elements by the establish-
ment of DNA methylation, are present in gymnosperms, but they
are highly specific to reproductive tissues and at substantially low-
er levels than in angiosperms (Nystedt et al., 2013). Furthermore, it
has been found that pseudogenes occur much more frequently
than functional protein-coding genes in the genome of gymno-
sperms (Kovach et al., 2010;Nystedt et al., 2013).
Many species of gymnosperms, especially conifers, have wide
distributions. Long generation time and large effective population
32 X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40

size are often hypothesized to be the causes for low synonymous
polymorphism in conifers (Savolainen and Pyhajarvi, 2007). Bus-
chiazzo et al. (2012) reported a slower evolutionary rate and a
higher d
N
/d
S
value in conifers than in angiosperms, and found a
d
N
/d
S
ratio >1 in many pair-wise comparisons of orthologs. How-
ever, Chen et al. (2012) found that the mean d
N
/d
S
value of gymno-
sperms is similar to that of angiosperms. Although they got a lower
synonymous substitution rate per year in gymnosperms, most of
this difference disappeared when generation time was taken into
account. It means that evolutionary constraints could be similar
between gymnosperms and angiosperms.
3.3.2.2. Chloroplast and mitochondrial genomes. Chloroplast and
mitochondrial genomes are predominantly maternally inherited
in angiosperms (Birky, 2008), except for a few species such as Si-
lene vulgaris (McCauley et al., 2005; reviewed by Crosby and Smith,
2012). However, the inheritance patterns of organelle genomes are
quite diverse in gymnosperms. The chloroplast genomes are pre-
dominantly paternally inherited in conifers but maternally inher-
ited in other gymnosperms, while the mitochondrial genomes are
maternally inherited in Pinaceae, Taxaceae, Cycadales and Gnetales
but paternally inherited in Araucariaceae, Cupressaceae s.l. and
Podocarpaceae (reviewed by Mogensen, 1996).
The chloroplast genome of most land plants consists of four
parts, including a large single copy (LSC) region, a small single copy
(SSC) region, and two copies of large inverted repeats (IRs) that
may be important for maintaining conserved gene orders (Palmer
and Thompson, 1982). Interestingly, different contraction of IRs
has been documented in gymnosperms (Raubeson and Jansen,
1992; Wu et al., 2011; Zhou et al., 2012). Ginkgo biloba has slightly
reduced IRs (Zhou et al., 2012). The extremely reduced IRs are
found in conifers (Lin et al., 2010; Wu et al., 2011), and the whole
chloroplast genome data suggest that Pinaceae and Conifer II have
lost different IR copies (Wu et al., 2011; Wu and Chaw, 2013).
However, gnetophytes have uncontracted IRs, although they have
the smallest compact chloroplast genomes in gymnosperms (Wu
et al., 2009). The occurrence of numerous structural rearrange-
ments in the chloroplast genomes of conifers strongly supports
the hypothesis that the reduction or deletion of IR could have made
the genome less stable (Hirao et al., 2008). It is particularly inter-
esting that the paternally inherited chloroplast genomes are signif-
icantly smaller than the maternally inherited ones except in
gnetophytes (Crosby and Smith, 2012). Therefore, it is likely that
the contraction of IRs is correlated with the shift to paternal inher-
itance of chloroplast in conifers. Furthermore, the ndh genes have
been lost from the chloroplast genomes of Pinaceae and gneto-
phytes, but still remain in conifer II (Braukmann et al., 2009; Wu
et al., 2009, 2011).
Compared to dozens of chloroplast genome sequences that are
available, the mitochondrial genome has been completely se-
quenced only for one gymnosperm, i.e., Cycas taitungensis, with a
size of 414.9 kb that is similar to the situation in angiosperms
(Chaw et al., 2008). The mitochondrial genome of cycad shows
many features different from that of angiosperms, including a
much higher proportion of tandem repeat (5%), a particularly
lower A + T content (53.1%), more RNA editing sites (1084) and
cis-spliced introns, and fewer gene losses (Chaw et al., 2008). How-
ever, it is still unclear whether these features are shared among
gymnosperms. For example, Regina et al. (2005) reported a novel
additional group II intron from the mitochondrial rps3 gene of Cy-
cas revoluta, and considered this intron as a signature of gymno-
sperms. However, Ran et al. (2010) and Regina and Quagliariello
(2010) found that this intron was lost in different lineages of gym-
nosperms. In particular, Ran et al. (2010) found a dramatically high
variation in both length and sequence of a rps3 exon in Conifer II
that could be attributed to the intron loss. In addition, Jaramillo-
Correa et al. (2013) reported an ancient microsatellite hotspot in
the conifer mitochondrial genome. They found that sequence tan-
dem repeats (STRs) can accumulate and be retained in short re-
gions of the mtDNA genome over long periods of time and
between distantly related lineages. It is unexpected that the draft
mitochondrial genome of Norway spruce is larger than 4 Mb, being
among the largest reported for plants and rich in short open-read-
ing frames (Nystedt et al., 2013). Therefore, more mitochondrial
genomes should be sequenced for understanding the evolutionary
pattern of mtDNA of gymnosperms.
4. Biogeography of gymnosperms
Historical biogeographic reconstruction relies largely on phylo-
genetic studies (Crisp et al., 2011; Yang et al., 2012). Extinction is a
key determinant of observed biogeographic patterns, but was often
considered intractable and ignored (Lamm and Redelings, 2009). In
recent years, developing synergies between phylogenetics, bioge-
ography, ecology, molecular dating and palaeontology have pro-
vided novel data and opportunities for testing biogeographic
hypotheses (Crisp et al., 2011; Ronquist and Sanmartín, 2011;
Gillespie et al., 2012; Mao et al., 2012; Yang et al., 2012; Wen
et al., 2013). The biogeography of gymnosperms has further been
studied with the help of new techniques and methods as well as
abundant microfossils and megafossils, shedding new light on
the origin and development of intercontinental disjunctions.
4.1. Disjunctive distribution in the two hemispheres
Some families and genera of gymnosperms are distributed in
both hemispheres (Table 1), which may provide an opportunity
to unveil the break-up history of Pangea. However, based on
molecular clock analysis, the crown ages of these genera, such as
Cycas,Ephedra,Gnetum and Podocarpus, could only be dated to
the Tertiary (mostly in or after the Oligocene), a time much later
than the separation between Gondwana and Laurasia (Won and
Renner, 2006; Ickert-Bond et al., 2009; Nagalingum et al., 2011;
Leslie et al., 2012). Therefore, their current distributions can only
be explained by long-distance dispersal among continents. It is
very likely that the genus Ephedra originated in Eurasia, then dis-
persed into North America in the Oligocene by the Bering Land
Bridge, and further into South America approximately 25 Mya, well
before the closure of the Panamanian Isthmus (Ickert-Bond et al.,
2009). In contrast, the genus Gnetum possibly originated in South
America, then dispersed by seawater to West Africa in the Oligo-
cene/Miocene, and finally to tropical and subtropical Asia, because
its seeds have special structures (Won and Renner, 2006). Although
a member of the family Taxaceae, Austrotaxus, is endemic to New
Caledonia, this monotypic genus separated from its sisters in the
Northern Hemisphere after the middle Cretaceous (Leslie et al.,
2012).
The coniferous family Cupressaceae s.l. does provide an excel-
lent example for studying the break-up history of Pangea. This
family occurs in all continents except Antarctica (Farjón, 2005),
and diversified into seven subfamilies during the Late Triassic
and Jurassic (Gadek et al., 2000; Mao et al., 2012; Yang et al.,
2012), predating or coinciding with the separation of Gondwana
and Laurasia. Two of the seven subfamilies, Athrotaxidoideae and
Callitroideae, are confined to the Southern Hemisphere. Consistent
with fossil evidence, the divergence between the Gondwanan
Callitroideae and its sister subfamily in the Northern Hemisphere,
Cupressoideae, could be dated back to the Jurassic (around 150–
180 Mya) by molecular clock analysis, providing strong evidence
for the vicariance between the two subfamilies by the spilt of
Laurasia and Gondwana (Li and Yang, 2002; Mao et al., 2012; Yang
X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40 33

et al., 2012). However, a relatively younger divergence between
Callitroideae and Cupressoideae (in the Cretaceous) was reported
in Crisp and Cook (2011) and Leslie et al. (2012). The subfamily
Athrotaxidoideae includes only one genus Athrotaxis, which com-
prises three extant species in Tasmania, with the most recent com-
mon ancestor dated to the Tertiary (Leslie et al., 2012; Mao et al.,
2012). However, fossils of this group have been reported from
North and South America in the lower Cretaceous sediments (Mill-
er and LaPasha, 1983; Del Fueyo et al., 2008), with an age younger
than the break-up of Pangea. Hence, the present distribution of
Athrotaxis could be the result of a southward expansion from North
America (Mao et al., 2012).
4.2. Disjunctive distribution in the Southern Hemisphere
Biogeographical patterns in the Southern Hemisphere have
drawn great interest from biologists and geologists (Sanmartin
and Ronquist, 2004; Knapp et al., 2005; Barker et al., 2007;
Upchurch, 2008; Yang et al., 2012), and were usually explained
by vicariance scenarios. However, recent studies suggest that these
patterns have also been shaped by dispersal events (McLoughlin,
2001; Givnish and Renner, 2004; Sanmartin and Ronquist, 2004;
Crisp et al., 2011). There are three lineages of conifers mainly dis-
tributed in the Southern Hemisphere, the subfamily Callitroideae
of Cupressaceae, Araucariaceae, and Podocarpaceae (Leslie et al.,
2012).
The Callitroideae is an ideal taxon for studying Southern Hemi-
sphere biogeography. It comprises 10 genera, each of which, except
Callitris and Libocedrus, is endemic to a single continent or even a
single island. These genera have relatively ancient origins (mostly
in the Cretaceous) and some reliable fossils (Mao et al., 2012; Yang
et al., 2012), although the divergence time estimates by Leslie et al.
(2012) are generally younger. Using multiple calibrations, Yang
et al. (2012) performed a relaxed molecular clock analysis for
Cupressaceae s.l. based on nuclear, chloroplast and combined gene
datasets, respectively, and reconstructed ancestral distributions for
the Callitroideae genera. They found that the separation of East and
West Gondwana at 165–130 Mya led to the divergence between
the two clades Callitris–Actinostrobus–Neocallitropsis and Widdring-
tonia–Fitzroya, and the split between Widdringtonia and Fitzroya–
Diselma that occurred at least 95 Mya as suggested by fossil evi-
dence is generally consistent with the final separation of Africa
from South America around 105 Mya (McLoughlin, 2001). There-
fore, vicariance could be mainly responsible for the current distri-
bution pattern of the Gondwanan Callitroideae. However, the other
two lineages, Araucariaceae and Podocarpaceae, have different bio-
geographic histories.
The Araucariaceae comprises three genera, Agathis,Araucaria
and Wollemia. Except two species of Araucaria (A. araucana and A.
angustifolia) in South America, all the other species of the family
are distributed in Australia and its adjacent regions (Farjón,
2010). Phylogenetic analysis and molecular dating indicate that
the two South American species diverged from their sisters Arau-
caria bidwillii in Australia and A. hunsteinii in New Guinea at Oligo-
cene or Miocene (Setoguchi et al., 1998; Leslie et al., 2012),
implying a dispersal event from Australia to South America be-
cause South America was connected with Australia through Ant-
arctica during 52–35 Mya (McLoughlin, 2001; Sanmartin and
Ronquist, 2004). Based on the phylogeny and divergence times of
Podocarpaceae (Knopf et al., 2012; Leslie et al., 2012), the dispersal
from Australia to South America through Antarctica could also
have occurred in three genera of the family, i.e., Lepidothamnus,
Podocarpus, and Prumnopitys. However, Saxegothaea, a monotypic
genus of Podocarpaceae in South America, originated around late
Jurassic to early Cretaceous (Leslie et al., 2012), and thus might
represent a remnant of ancient lineages. Although Podocarpus is
widely distributed in Asia, South America, Africa, and Australia
and its neighboring islands, this genus and its main lineages di-
verged in the Paleogene and Neogene (Biffin et al., 2011; Leslie
et al., 2012). Therefore, its present wide distribution could also
be attributed to long-distance dispersal.
4.3. Disjunctive distribution in the Northern Hemisphere
The biogeographic patterns in the Northern Hemisphere are
complex due to effects of both vicariance and frequent dispersal
by the Bering Land Bridge (BLB) and the North Atlantic Land Bridge
(NALB), and the Eastern Asian-North American disjunction is par-
ticularly interesting (Ian Milne, 2006; Wen, 1999). Previous studies
suggest that the Asia-to-New World migration was common for
angiosperm lineages (Wen, 1999; Wen et al., 2010). However, cur-
rent data imply that more gymnosperm lineages could have expe-
rienced a history of the New World-to-Old World migration (Ran
et al., 2006; Wei et al., 2010; Wen et al., 2010).
All the thirteen gymnospermous genera that are disjunctly dis-
tributed between North America and Eurasia or Asia (Table 1,
Cupressus is not included) dispersed by the BLB one or more times
during their evolutionary history (Fig. 5). Seven of them likely orig-
inated in North America and migrated into the Old World, includ-
ing Abies (Xiang et al., 2009), Chamaecyparis (Wang et al., 2003),
Larix (Wei and Wang, 2003), Picea (Ran et al., 2006; Klymiuk and
Stockey, 2012), Pseudotsuga (Wei et al., 2010), Taxus (Li et al.,
2001; Hao et al., 2008), and Thuja (Peng and Wang, 2008). In con-
trast, an Eurasian origin and dispersal to North America were in-
ferred for only four genera, including Ephedra (Ickert-Bond et al.,
2009), Juniperus (Mao et al., 2010), Pinus (Eckert and Hall, 2006;
Ryberg et al., 2012), and Torreya (Hao et al., 2008). The biogeo-
graphic history of the rest two genera, Calocedrus and Tsuga, has
not been well resolved, although vicariance between East Asia
and North America was suggested for them (Chen et al., 2009; Hav-
ill et al., 2008).
According to the molecular phylogeny of extant species, Tsuga
could have a North American origin. However, likelihood-based
biogeographic inference using Lagrange, which incorporated phy-
logeny, divergence times, fossil data, and rates of lineage dispersal
and extinction, inferred an Eocene basal crown group diversifica-
tion and an initial widespread circumpolar distribution with sub-
sequent vicariance and extinction events for the genus (Havill
et al., 2008). Lockwood et al. (2013) reported a molecular phylog-
eny of Picea, and suggested an Asian origin for the genus based
mainly on signal from large motifs in mitochondrial gene introns.
It should be reminded that this kind of introns often shows intra-
specific variation in both sequence and structure, and thus is not
suitable for the reconstruction of interspecific relationships if only
a couple of individuals are sampled for each species. Based on our
recent studies (unpublished data), all cytoplasmic and nuclear
gene trees still support the basal position of the western North
American Picea breweriana and a North American origin of the
genus. Our results are also consistent with the fossil record (Le-
Page, 2001), especially the earliest fossil of Picea from northern
Vancouver Island dated to 136 Ma (Klymiuk and Stockey, 2012).
Some lineages of Juniperus and Pinus possibly migrated from
Eurasia to North America by the NALB (Eckert and Hall, 2006;
Mao et al., 2010). Moreover, multiple dispersal events between
Eurasia and North America could have occurred in Pinus (Eckert
and Hall, 2006), Picea (Ran et al., 2006), and Juniperus (Mao et al.,
2010). Although some genera that originated in Eurasia, such as
Juniperus and Ephedra, have a distribution in high latitudes and al-
pine regions, their ancestors possibly occurred in warmer habitats
and might represent remnants of the Madrean-Tethyan vegetation
belts (Eckert and Hall, 2006; Ickert-Bond et al., 2009; Mao et al.,
2010).
34 X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40

4.4. Biogeographic difference between the two hemispheres
As summarized above, both vicariance and dispersal events
played important roles in shaping the present distribution patterns
of the Southern Hemisphere conifers while dispersal contributed
much more than vicariance in developing intercontinental disjunc-
tions of gymnosperms in the Northern Hemisphere. The direction
of historical intercontinental dispersal is also different between
the Southern and Northern Hemisphere gymnosperms. Simply,
dispersal in the Southern Hemisphere shows an ‘‘out-of-Australia’’
mode, but that in the Northern Hemisphere can be generalized as
‘‘bidirectional’’, i.e., ‘‘out-of-America’’ and ‘‘out-of-Eurasia’’.
For the distribution pattern of gymnosperms, most genera
mainly distributed in the Southern Hemisphere differ greatly from
the genera in the Northern Hemisphere. A total of 41 and 43 genera
are distributed (or mainly distributed) in the Southern Hemisphere
and the Northern Hemisphere, respectively, of which 15 occur
widely in both Eurasia and North America while only eight live
in more than one continents in the Southern Hemisphere (Table 1).
That is, the Southern Hemisphere genera are more isolated than
the North Hemisphere ones. This distribution difference could be
caused by the fundamental difference between the two hemi-
spheres in geography. The three main continents, Africa, South
America and Australia, separated at least 83 Mya, preventing plant
dispersal among the continents in the Southern Hemisphere.
Although South America was connected to Australia through Ant-
arctica during 52–35 Mya (McLoughlin, 2001; Sanmartin and Ron-
quist, 2004), only some cold-tolerant plants could spread by this
land bridge (Hill and Brodribb, 1999). Unfortunately, most of the
gymnosperm lineages in the Southern Hemisphere prefer rela-
tively wet and warm environments (Hill and Brodribb, 1999; Leslie
et al., 2012; Pittermann et al., 2012), and could not disperse by
Antarctic. They might tend to expand into some wet and warm
places, like the ten genera that have a Southern Hemisphere origin
but now occur in the tropic areas of both Australia and Asia
(Table 1; and Fig. 5). On the contrary, due to the climatic cooling
in the Cenozoic, especially the extensive glaciations, the middle
and high-latitude land masses in the Northern Hemisphere shifted
to colder, drier, and more seasonal climates from warm subtropical
and temperate climates since the Oligocene (Wolfe, 1994; Ivany
et al., 2000; Zachos et al., 2001; Moran et al., 2006; Dupont-Nivet
et al., 2007; Zanazzi et al., 2007; Eldrett et al., 2009). Some lineages
that better adapted to cooler and/or drier conditions could have re-
placed older lineages. Furthermore, Beringia was not covered by
ice during Quaternary glaciations, and could have played dual roles
as both a glacial refugium and a route of colonization of plants
across the continents of Eurasia and North America (Hopkins,
1967). Therefore, repeated instances of species migration, and
range contraction and expansion responding to glacial cycles could
also have contributed to the complex biogeographic patterns of the
Northern Hemisphere gymnosperms (Leslie et al., 2012).
Cycads are distributed in most subtropical and tropical regions
of the world. However, except that Cycas, the biggest genus of cy-
cads, has a relatively wide distribution in Asia, Australia and Africa,
all the other genera are confined to a single continent or two con-
tinents once connected, such as Zamia in the tropical areas of South
America and North America. Molecular dating indicates that
Cycadaceae and Zamiaceae diverged from each other in the Jurassic
and all genera of the two families originated at least before Oligo-
cene (Nagalingum et al., 2011). According to fossil evidence, cycads
were diverse in the Mesozoic, but experienced extinctions toward
the end of the Mesozoic (Norstog and Nicholls, 1997; Hermsen
et al., 2006; Taylor et al., 2009). This group underwent a nearly
synchronous global species rediversification that began in the late
Miocene, followed by a slowdown toward the recent (Nagalingum
et al., 2011). During the secondary species diversification, why did
the distribution of the genera not expand largely? It would be
interest to investigate the speciation of cycads, which may provide
answers for why all species of this group are endemic and
endangered.
5. Concluding remarks
In the past two decades, there have been exciting advances in
our understanding of gymnosperm evolution and biogeography,
including reconstructed phylogenetic relationships at different
taxonomic levels based on comprehensive evidence, patterns of
species diversification such as recent radiation in most lineages,
relative roles of vicariance and dispersal in development of
Fig. 5. Inferred dispersal routes of some gymnospermous genera with intercontinental distributions. BLB, Bering Land Bridge; NALB, North Atlantic Land Bridge; AF, Africa;
AU, Australia (continent); EA, Eurasia; NA, North America; SA, South America. The question marks indicate migration routes of the taxa have not been resolved, while the dash
arrow indicates a possible route of migration.
X.-Q. Wang, J.-H. Ran / Molecular Phylogenetics and Evolution 75 (2014) 24–40 35

intercontinental disjunctions, modes of molecular evolution in dif-
ferent genomes and groups, and mechanisms underlying the for-
mation of large nuclear genome. In particular, most evidence
supports the Gnepine hypothesis; Multiple coniferous genera such
as Picea very likely originated in North America and migrated into
the Old World, further indicating that the center of diversity is not
necessarily the place of origin; Conifers have very similar numbers
of unigenes as angiosperms in the nuclear genome, and expansion
of retrotransposons is responsible for their large and complex gen-
omes. However, the Gnepine hypothesis has not been completely
accepted by botanists, and it is still difficult to understand the sis-
ter relationship between Gnetales and Pinaceae from morphologi-
cal characters. In addition, the evolutionary relationships of
congeneric species that originated from recent radiation are diffi-
cult to be resolved due to frequent interspecific gene flow and
incomplete lineage sorting. The molecular phylogenies with low
resolution should be cautiously used in biogeographic inference.
It also should be mentioned that most fossil evidence used in pre-
vious biogeographic studies of the Northern Hemisphere gymno-
sperms is from Europe and North America. More fossil evidence
from Asia is particularly helpful to test the biogeographic
hypotheses.
The draft genome sequences of conifers provide an unprece-
dented opportunity and an important platform for the evolution-
ary studies of gymnosperms, especially for unraveling the
mechanisms of genome evolution and the genetic basis of morpho-
logical characters. It will also shed new light on evolution of many
important gene families and biological pathways in seed plants,
even land plants. However, these genome data should be carefully
used in phylogenetic reconstruction of gymnosperms before the
gene orthology/paralogy is clarified. According to our experience,
using some genes with a clear evolutionary history is much better
than using all genes in the genome that have not been well studied.
Phylogenomics does not always work very well. Moreover, it
would be very valuable to investigate the speciation mechanisms,
the adaptive value of large nuclear genomes, and the response of
genome to environmental changes in gymnosperms using next-
generation sequencing techniques. Also, more phylogeographical
studies are encouraged to reveal the response of gymnosperms to
climatic oscillations and the locations of glacial refugia, which need
a wide population sampling of closely related species. This kind of
knowledge is important for the conservation of gymnosperms.
Acknowledgments
The authors thank the anonymous reviewer’s insightful com-
ments and suggestions on the manuscript. We also thank Dr.
Dong-Mei Guo for providing some information of Gnetales. This
work was supported by the National Natural Science Foundation
of China (Grant Numbers: 31170197, 31330008).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2014.02.
005.
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