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Rediscovery, discrimination and environmental correlates of Dianella species 35
Rediscovery of the endemic gastropod
Dianella schlickumi (Gastropoda, Hydrobiidae) and its
discrimination from Dianella thiesseana: environmental
correlates and implications for their conservation
Chrysoula Ntislidou1,2, Canella Radea3, Sinos Giokas4, Martin T. Pusch2,
MariaLazaridou1, Dimitra C. Bobori1
1 Department of Zoology, School of Biology, Aristotle University of essaloniki, 54124 essaloniki, Greece
2Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), 12587 Berlin, Germany 3 Department
of Ecology and Systematics, Faculty of Biology, School of Sciences, National and Kapodistrian University of
Athens, 15784, Panepistimioupolis, Greece 4 Department of Biology, University of Patras, 26500, Patra, Greece
Corresponding author: Chrysoula Ntislidou (ntislidou@bio.auth.gr)
Academic editor: M. Pinna |Received 7 January 2018 | Accepted 24 May 2018 |Published 11 June 2018
http://zoobank.org/EF862219-884C-436F-BF54-7603F97EEFFB
Citation: Ntislidou C, Radea C, Giokas S, Pusch MT, Lazaridou M, Bobori DC (2018) Rediscovery of the endemic
gastropod Dianella schlickumi (Gastropoda, Hydrobiidae) and its discrimination from Dianella thiesseana: environmental
correlates and implications for their conservation. Nature Conservation 27: 35–58. https://doi.org/10.3897/
natureconservation.27.23289
Abstract
e aquatic snail genus Dianella (Gastropoda: Hydrobiidae) has only two representatives in Greece: Di-
anella schlickumi Schütt, 1962 and Dianella thiesseana (Kobelt, 1878). D. schlickumi, a narrow endemic
species to Lake Amvrakia (in Aitoloakarnania, western-central Greece), is considered as Critically Endan-
gered (Possibly Extinct, sensu IUCN 2017). Our study conrmed its presence in Lake Amvrakia, where
it had not been detected for more than 30 years. We document the unknown anatomical characters based
on the D. schlickumi specimens. Moreover, the presence of D. thiesseana in the nearby lakes Trichonis and
Lysimachia was also conrmed, while morphometric analyses enabled the discrimination between the two
species. Redundancy Analysis revealed conductivity, dissolved oxygen and pH as the main environmental
variables related to the above species’ distribution, shaping their community structure. Both Dianella spe-
cies require urgent conservation measures to be enforced, due to their habitat degradation from human
activities, which are limiting and fragmenting their range. For that purpose, eective management plans
have to be elaborated and implemented at the mentioned lakes, focusing on the reduction of human pres-
sures and on the improvement of their habitats.
Nature Conservation 27: 35–58 (2018)
doi: 10.3897/natureconservation.27.23289
http://natureconservation.pensoft.net
Copyright Chrysoula Ntislidou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
36
Keywords
Pyrgulinid, Greek lakes, anatomy, morphometrics, environmental correlates, conservation
Introduction
Freshwater habitats cover less than 1% of the earth’s surface, but they support almost
10% of the known species on the planet (Strayer and Dudgeon 2010). However, fresh-
water species are in a worldwide decline (Howard et al. 2015), resulting thus in the
decrease of freshwater biodiversity, which is mainly threatened by over-exploitation,
water pollution, hydrological alterations, destruction and degradation of habitats and
invasion by exotic species (Dudgeon et al. 2006).
e Balkan region is rich in freshwater fauna due to high endemism (Grith et
al. 2004, Glöer et al. 2007). e rich fauna of freshwater molluscs is considerably
threatened (Albrecht and Wilke 2008) and 29 species of the Balkan Peninsula have
already become extinct (Régnier et al. 2009), representing 21% of the 140 known
extinctions of freshwater mollusc species worldwide. In this region, many endemic
gastropod species exist with a restricted range only extending to small hydrographic
systems (rivers, lakes and springs). e karst landscape, which occurs in a large pro-
portion of the Balkan Peninsula, also contributes to this high degree of endemism
(Régnier et al. 2009). ese species are vulnerable not only due to habitat degrada-
tion caused by human activities, but also by their very limited range, which increases
the risk of accidental extinction (Régnier et al. 2009). For Greece, a similar crucial
decline of population densities and losses of some endemic mollusc species have
been reported (Albrecht et al. 2006), suggesting the necessity for urgent actions for
their conservation.
e distribution of gastropod species is often associated with biotic and abiotic
habitat parameters (e.g. Covich 2010), which are related to specic geographical char-
acteristics, physicochemical conditions, biological interactions and historical and ran-
dom factors (Lodge et al. 1987). us, the distributional patterns of endemics can be
often better explained based on records of key environmental parameters, which drive
the structure of biological communities (Pérez-Quintero 2012). Such knowledge may
also help to identify and protect specic regions for the conservation of biodiversity
of little-known freshwater bodies (Abell et al. 2007), especially in the Mediterranean
region which has been neglected.
e species of the subfamily Pyrgulinae (Caenogastropoda: Truncatelloidea: Hyd-
robiidae) are widely distributed in western Asia as well as in central and south-eastern
Europe (Wilke et al. 2007). In the latter area, the subfamily consists of ten genera,
namely Chilopyrgula Brusina, 1896, Dianella Gude, 1913, Ginaia Brusina, 1896, Neo-
fossarulus Polinski, 1929, Ochridopyrgula Radoman, 1955, Pyrgohydrobia Radoman,
1955, Pyrgula De Cristofori & Jan, 1832, Stankovicia Polinski 1929, Trachyochridia
Polinski, 1929 and Xestopyrgula Polinski, 1929, comprising 17 species (Bank2017).
Rediscovery, discrimination and environmental correlates of Dianella species 37
e pyrgulinids are mostly lacustrine snails, colonising the littoral and sublittoral zones
of ancient deep freshwater lakes (Radoman 1983, Wilke et al. 2007, Schreiber et al.
2012). Only one species, Chilopyrgula sturanyi Brusina, 1896, has been recorded in
springs (Radoman 1983).
Out of those, two pyrgulinid species, belonging to the genus Dianella Gude, 1913,
are known to be present in lentic systems of Greece: Dianella schlickumi Schütt, 1962
and Dianella thiesseana (Kobelt, 1878). e rst species is known from Lake Amvrakia
and the latter from lakes Trichonis and Lysimachia (Aitoloakarnania, western-central
Greece) (Schütt 1962, Radoman 1973, 1983, Szarowska et al. 2005, Szarowska 2006).
Both species are narrow endemic species, with major gaps in historical records, while
no information is available about their abundance, population structure, life cycle or
genetic diversity. In the IUCN Red List of reatened Species, Dianella thiesseana
appears under the status “Critically Endangered”, while D. schlickumi has been de-
clared as “Possibly Extinct” (IUCN 2017). Apart from Dianella, the aquatic gastropod
communities of these lakes include several other endemic hydrobiids, such as Islamia
graeca Radoman, 1973, Islamia trichoniana Radoman, 1979, Pseudoislamia balcanica
Radoman, 1979, Trichonia trichonica Radoman, 1973 (Radoman 1983). D. schlickumi
has not been observed for more than 30 years, despite several sampling eorts per-
formed in Lake Amvrakia (Reischütz and Reischütz 2002, Szarowska et al. 2005, Al-
brecht et al. 2006, 2011a), which is considered as the type locality of this species. Only
fresh empty shells of this species were recorded in an irrigation channel, near Acheloos
River (close to Lake Amvrakia) by Reischütz et al. (2008). D. thiesseana has only been
found at the northeast bank of Lake Trichonis during the last decade (Albrecht et al.
2009, 2011b).
e external morphology, the anatomy and the phylogenetic position of the
majority of Pyrgulinae have been extensively studied and discussed (Schütt 1962,
Radoman 1983, Riedel et al. 2001, Szarowska et al. 2005, Szarowska 2006, Anis-
tratenko 2008, Wilke et al. 2013). e combination of anatomical and phylogenetic
data suggests that the Pyrgulinae subfamily has a very close (possibly even sister-
group) systematic relationship with Hydrobiinae (Szarowska et al. 2005). Concern-
ing the two species of Dianella, it has been reported that they have similar exter-
nal morphology and soft body anatomy, but dierent body size (Radoman 1983).
However, the inter-specic dierences have been questioned by Szarowska et al.
(2005), which has made the taxonomic distinction between D. thiesseana and D.
schlickumi unclear and questionable. Although the soft body anatomy of D. thies-
seana is described and/or depicted in detail (Radoman 1983, Szarowska et al. 2005,
Szarowska 2006), various anatomical characters of D. schlickumi remain unknown,
un-described or unpublished.
Hence, the aims of the present study were to: (a) report the rediscovery of the
seemingly extinct pyrgulinid D. schlickumi, (b) describe its unknown anatomical char-
acters, (c) examine its morphometric discrimination from D. thiesseana and (d) reveal
the environmental parameters that probably drive the distribution of both species.
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
38
Material and methods
Study area
We studied three natural lakes located in the western-central part of Greece (Figure1).
ese lakes are considered as the last remains of a former big lake, which by several
processes, has been split into four smaller lakes (Albanakis et al. 1995). e Acheloos
River separates Amvrakia and Ozeros lakes from Trichonis and Lysimachia lakes (Fig-
ure 1). Lake Amvrakia (Table 1) is a semipolje karst lake of tectonic origin with high
sulphate concentrations, situated in Mesozoic limestone (Verginis and Leontaris 1978,
Overbeck et al. 1982). Its water level shows signicant seasonal uctuations, which
may be attributed to various factors such as the hydraulic communication with karst
aquifers, high evaporation rates, especially during summer and intensive use of water
for agricultural purposes (Danielidis et al. 1996). Lake Trichonis (Table 1) is the deep-
est and the largest natural lake in Greece, which is also a karst water body (Zacharias
et al. 2002), situated in a tectonically active area (Poulimenos and Doutsos 1997). It is
supplied by precipitation, approximately 30 intermittent streams and sub-aquatic karst
springs (Dimitriou and Zacharias 2006). At its western end, a narrow canal connects
Lake Trichonis with Lake Lysimachia (Table 1), which is shallower (Figure 1) and it
was receiving the untreated urban wastewaters from the city of Agrinio until the year
2000. At its western end, Lake Lysimachia drains to the Acheloos River via an articial
canal (Figure 1).
Field sampling
Macroinvertebrate samplings were conducted in spring and autumn 2014 by boat, at
the sublittoral and profundal zones of each lake, using an Ekman-Birge grab (three
replicates per station, 225 cm2 sampling area). A total of 66 samples were collected in
the studied lakes [Amvrakia: 14 stations (7 stations at each zone), Trichonis: 13 (5 and
7 stations at the sublittoral and profundal zones, respectively) and Lysimachia: 6 (3 sta-
tions at each zone); Figure 1]. Samples were sieved with a 500 μm mesh and preserved
Table 1. Geographical position, morphological and limnological features of the studied lakes. Alt: al-
titude; Zmean: mean depth; Zmax: max depth; La: lake surface area; TL: trophic level, ET: eutrophic, MT:
mesotrophic, OL: oligotrophic; WMM: warm monomictic.
Lake Latitude Longititude Alt (m.a.s.l) Zmean
(m) Zmax
(m) La (km2) TL Lake
type
Trichonis 38.573333 21.552222 18†30†57‡96.5 OL/MT§WMM†
Lysimachia 38.558597 21.376873 16†3|9|13.1 ET | WMM|
Amvrakia 38.652150 21.219867 16†23 53¶11.8 MT#WMM#
†Zacharias et al. (2002), ‡Skoulikidis et al. (1998), §Doulka and Kehayias (2008), |Leonardos (2004), ¶Albanakis et al.
(1995), #Danielidis et al. (1996)
Rediscovery, discrimination and environmental correlates of Dianella species 39
in 70% ethanol. After sorting, zoobenthos was identied to the lowest possible taxon
and abundance was converted to density (ind./m2). Additionally, at each station, water
samples from 1 m above the bottom of each lake were collected, using a Niskin-type
sampler. Environmental parameters such as water temperature (WT, 0C), pH, conduc-
Figure 1. Geographical location of the studied lakes and sampling stations.
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
40
tivity (Cond, μS/cm) and dissolved oxygen concentration (DO, mg/l) were measured
in situ just above the bottom sediment of each station using the Aqua Read AP-2000
probe (Kent, GB). Secchi depth (Secchi, cm) was also recorded.
Anatomy
Genitalia of male and female specimens and other soft body characteristics of D. schlic-
kumi from Lake Amvrakia were studied and compared with those of D. thiesseana
collected from Lake Trichonis during the present study and with some additional in-
dividuals collected in 2016 (Figure 2). Before dissection, the shells of the specimens
studied were removed by soaking in Perenyi solution, following Hershler and Ponder’s
(1998) morphological terminology. e characterisation of the nervous system was
based on Davis et al. (1986) and its concentration was measured as the RPG ratio
(Davis et al. 1976).
Morphometry
Shell morphometry was recorded in both D. thiesseana (Trichonis: 33 individuals and
Lysimachia: 5 individuals) and D. schlickumi (Amvrakia: 44 individuals) in well pre-
served shells of adult specimens, using the software ImageFocus v3.0.0.1. Seven linear
measurements were taken, specically: shell height (H), shell width (W), shell aperture
height (Ha), shell aperture width (Wa), spire height (SH), body whorl height (BWH)
and penultimate whorl height (PWH) (Figure 3). Measurements were then expressed
as ratios: shell height/shell width (H/W); shell aperture height/shell aperture width
(Ha/Wa); spire height/body whorl height (SH/BWH), penultimate whorl height/body
whorl height (PWH/BWH) and body whorl height/shell width (BWH/W). Prior to
further analyses, all the linear measurements and the ratios were logarithmically trans-
formed for normalisation. Subsequently, the t-test for independent samples was per-
formed for detecting signicant dierences (p < 0.05) between the two species using
the statistical software SPSS v22.
e studied populations were also tested for morphological diversication using
a landmark-based method of acquiring geometrical data of shape and size. Shell var-
iation has been traditionally quantied through linear measurements and ratios to
distinguish between individuals and populations, amongst and within snail species.
Recently, geometric morphometrics (GMs) have been employed for examining shells,
both to provide direct size-free analyses of shell shape (Conde-Padin et al. 2007b,
Hayes et al. 2007) and to answer broader evolutionary questions (Pfenninger and
Magnin 2001, Conde-Padin et al. 2007a, Haase and Misof 2009, Páll-Gergely et al.
2012, Giokas et al. 2014). GMs have the advantage over traditional morphometrics in
being, theoretically, free of eects due to size, position, rotation and scale (Rohlf and
Marcus 1993).
Rediscovery, discrimination and environmental correlates of Dianella species 41
For that purpose, we used sub-samples of the undamaged and well preserved shells
of adult specimens (i.e. 15 D. schlickumi and 9 D. thiesseana specimens), using the
aperture features. Specimens were set down on the same plane, with the aperture fac-
ing up and we took digital photographs of them. Geometric morphometric variables
of the shells were obtained with 18 landmarks (LM) representing the outline of the
shell and of the aperture as shown in Figure 4. Landmarks were digitised using tpsDig
(Rohlf 2006) and occupied the same positions over the sum of the specimens. Two of
these (LM1 and LM18) were of type I (developmentally homologous), 12 landmarks
(LM2-LM13) represented suture lines and were considered type II (geometrically ho-
mologous). e remaining landmarks were either extreme points on surfaces or were
placed halfway between other landmarks, making them type III (Bookstein 1991,
Zelditch et al. 2004).
All geometric morphometric analyses were performed with MorphoJ (Klingen-
berg 2011). e coordinates of shape to be used for further statistical analysis were
obtained with Procrustes generalised least squares superimposition. is method ex-
cludes the impact of size on the shape of the shell and, theoretically, variations inde-
pendent of shape are removed using this analysis (Zelditch et al. 2004). Size variables
Figure 2. Specimens of: Dianella schlickumi from Lake Amvrakia (A–E) and Dianella thiesseana from
Lake Trichonis (F–J), demonstrating their large inter-specic variation of shell morphology.
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
42
Figure 3. External morphological measurements taken in specimens of Dianella schlickumi and Dianella
thiesseana.
Figure 4. Dianella schlickumi and Dianella thiesseana specimens positioned with the axis of the shell on
the y-axis and the aperture in the same plane as the objective, showing the location of the 18 landmarks
(LM1-LM18) used. A D. schlickumi B D. thiesseana.
Rediscovery, discrimination and environmental correlates of Dianella species 43
(centroid size) for each specimen were also generated. We examined, using pooled
within-group regression analysis, the eect of centroid size on shape, regressing to the
resulting Procrustes coordinates on centroid size. at regression score was not signi-
cant (Permutation test, 10,000 rounds against the null hypothesis of independence, %
predicted = 6.170, p = 0.2183). erefore, we eventually used the Procrustes coordi-
nates, which were analysed with Principal Component Analysis (PCA) and Canonical
Variate Analysis (CVA) in order to explore the morphological variation between the
two Dianella species. e reliability of the discrimination was assessed by leave-one-
out cross-validation.
Analyses of abundance and environmental data
Detrended Correspondence Analysis (DCA) (indirect gradient analysis) (programme
CANOCO version 4.5.1; Ter Braak and Šmilauer 1998) was performed to abundance
data in order to verify if they corresponded linearly to the environmental gradient or
if abundances peaked around an environmental optimum (unimodal response) (Ter
Braak and Šmilauer 1998). In this study, the species abundance data corresponded
roughly linearly to the gradient, because the length of the gradient of the rst axis was
three times less than the range of the within-sample standard deviation (Ter Braak and
Šmilauer 1998). us, Redundancy Analysis (RDA) was further applied to link the en-
vironmental parameters to species abundance data. e Monte Carlo permutation test
was performed to select the statistically signicant (p <0.05) environmental parameters
that explain most of the variance of species abundance, while the variation ination
factor (>20) was used to assess the interrelation of the parameters.
Results
Species distribution
Dianella schlickumi was found at the south-east part of Lake Amvrakia, in 5 (S7, S11,
S12, S13, S14; Figure 1) out of the 14 stations (36%) surveyed, at depths ranging from
5 m to 13 m. No individuals were recorded at its western part (S9 and S10, Figure 1),
not even empty shells. e highest abundance was recorded at station S15 (607 ind./
m2) at depth 5 m (Table 2). D. thiesseana was found in 2 (S6 and S13) out of the 13
stations (15%) in Lake Trichonis, located on the northeast side of the lake, at depths
of 13 m to 18 m (Figure 1, Table 2). During the current study, only empty shells from
D. thiesseana were collected in Lake Lysimachia. However, the presence of a live D.
thiesseana was conrmed in the east side of Lake Lysimachia by surveys executed dur-
ing the National Water Monitoring Programme in spring 2015 (Mavromati, personal
communication). Both species were present during the spring surveys while none was
found during autumn.
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
44
Table 2. Dianella schlickumi and Dianella thiesseana abundances (number of individuals sampled by
Ekman-Birge grab, 225 cm2 sampling area) per sampling station and environmental parameters measured
in lakes Trichonis, Lysimachia and Amvrakia.
Lake Station
Abundance (ind./m2) Environmental parameters
Dianella
schlickumi
Dianella
thiesseana
Depth
(m)
Secchi
(m)
DO
(mg/l) pH Cond
(μS/cm)
WT
(oC)
Trichonis S6 59 18.0 6.4 9.05 10.04 319 18.8
S13 44 13.0 5.6 8.92 10.73 320 15.0
Lysimachia LYS 15 3.8 1.7 9.77 8.13 363 11.9
Amvrakia
S7 15 13.0 5.2 8.87 9.74 982 21.2
S11 15 8.0 4.7 8.38 10.10 921 20.5
S12 44 11.5 4.8 8.48 10.48 922 20.5
S13 44 9.0 5.1 8.66 10.42 916 21.6
S14 607 5.0 3.2 8.47 10.62 898 20.9
Soft body anatomy of D. schlickumi
Ctenidium-Osphradium: Ctenidial laments broader than high; osphradium elon-
gate, approximately opposite to the middle of ctenidium.
Nervous system (Figure 5A): Cerebral ganglia similarly sized, white; supraoesophageal
and suboesophageal ganglia white; supraoesophageal connective much longer than
the suboesophageal. Nervous system extremely elongate, RPG ratio 0.75.
Gastric caecum: Large and elongate gastric caecum on the posterior stomach chamber.
Rectum: e U-shaped intestine loop in the pallial cavity roof was wide and the faecal
pellets were packed sideways (Figure 5B).
Female reproductive system (Figure 6A): Pallial oviduct glands with straight border;
bursa copulatrix medium-sized, ovoid-globular, positioned posteriorly to the albu-
men gland; bursal duct long; renal oviduct unpigmented, coiled tightly with more
than two bends; typical seminal receptacles absent; a beak-shaped dilatation of
oviduct in the position of seminal receptacle rs2, anteriorly to the bursa.
Penis (Figure 6B): Penis large, gradually tapered, proximal part folded, penial base of
medium width, its attachment area central and well behind the head, penial duct
almost straight located at the outer edge of the penis.
Egg capsule (Figure 6C–D): Several lenticular and transparent egg capsules (each one
containing a single egg) in various growth stages were found on the external shell
surface of two specimens.
Morphometry
Seven out of the 12 morphometric measurements taken, diered statistically (p
< 0.001) between the two species. Generally, individuals of D. thiesseana exhibited
higher mean values compared to D. schlickumi, except for SH/BWH ratio, which was
higher in D. schlickumi (Table 3).
Rediscovery, discrimination and environmental correlates of Dianella species 45
Table 3. Average (±standard error) and range (minimum and maximum values) of the morphometric
measurements (in mm) taken in Dianella thiesseana and Dianella schlickumi specimens. e results of
t-test are also provided (* indicates the signicant dierences). n = number of individuals used: H: shell
height; W: shell width; Ha: shell aperture height; Wa: shell aperture width; SH: spire height; BWH: body
whorl height; PWH: penultimate whorl height.
Variables Dianella thiesseana
(n=38)
Dianella schlickumi
(n=44)
t-test
F p
H 6.79 ± 0.25 (4.16–9.78) 6.17 ± 0.21 (4.34–10.89) 3.95 0.063
W* 3.01 ± 0.07 (2.20–3.86) 2.65 ± 0.06 (1.93–3.75) 0.40 <0.001
Ha* 2.45 ± 0.06 (1.58–3.16) 2.07 ± 0.04 (1.61–2.92) 1.45 <0.001
Wa* 1.67 ± 0.04 (1.18–2.22) 1.45 ± 0.03 (1.05–1.91) 0.04 <0.001
SH 4.10 ± 0.19 (2.05–6.38) 3.97 ± 0.17 (2.50–7.70) 8.87 0.797
BWH* 2.69 ± 0.07 (2.00–3.77) 2.20 ± 0.05 (1.64–3.25) 0.86 <0.001
PWH* 0.67 ± 0.02 (0.34–0.92) 0.54 ± 0.01 (0.34–0.78) 5.23 <0.001
H/W 2.24 ± 0.06 (1.69–3.14) 2.31 ± 0.04 (1.99–3.29) 3.09 0.250
Ha/Wa 1.47 ± 0.02 (1.19–1.82) 1.44 ± 0.02 (1.16–1.93) 0.85 0.255
SH/BWH* 1.52 ± 0.06 (0.87–2.37) 1.80 ± 0.04 (1.36–2.89) 12.14 <0.001
PWH/BWH 0.25 ± 0.01 (0.16–0.38) 0.25 ± 0.00 (0.17–0.32) 0.62 0.731
BWH/W* 0.89 ± 0.01 (0.77–1.09) 0.83 ± 0.01 (0.73–0.98) 0.15 <0.001
Figure 5. Non-genital anatomy of Dianella schlickumi. A Nervous system B Rectum full of faecal
pellets. Abbreviations: cm -commissure; fp -faecal pellets; lc -left cerebral ganglion; lp -left pleural
ganglion; sb - suboesophageal ganglion; sp -supraoesophageal ganglion; r -rectum.
Geometric morphometric analysis revealed a clear size and shape distinction between
the two Dianella species. Shells of D. thiesseana were signicantly larger (Centroid Size)
than those of D. schlickumi (F1, 22 = 38.01, p < 0.0001, Figure 8). e landmark-based
analyses of individual shells also conrmed a shape distinction between the two species.
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
46
Figure 6. Genital anatomy and egg capsules of Dianella schlickumi. A Female genitalia B Penis C Shell
bearing egg capsules covered by ne-grained material D Egg capsule with embryo. Abbreviations: ag–
albumen gland; b–beak-shape dilatation of the oviduct; bc–bursa copulatrix; fp–faecal pellets; fg–ne-
grained material; e–eye; ec–egg capsules; em–embryo; ov: renal oviduct; pd–penial duct.
ANOVA of Procrustes coordinates also showed a clear shape dierence between the two
species (F32, 704 = 20.78, p < 0.0001). PCA of Procrustes coordinates, which aimed at nd-
ing linear combinations maximising the total variance, revealed three principal components
(PC1-PC3) explaining 88.1% of the total variance. e distinction between the two species
was quite evident along the PC1 axis (Figure 9). In the CVA, which maximised dierentia-
tion amongst species (or predened groups), the rst and only extracted canonical variate
Rediscovery, discrimination and environmental correlates of Dianella species 47
Figure 7. RDA ordination diagram of Dianella thiesseana and Dianella schlickumi abundances at sam-
pling sites (circles) in relation to environmental parameters in lakes Trichonis (TRI), Lysimachia (LYS)
and Amvrakia (AMV) in spring 2014 and 2015.
Figure 8. Means ± 95% Condence Intervals of centroid size for the two Dianella species and their
ordinations based on landmark data.
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
48
(CV1) explained 100% of total variation and showed a much clearer separation than PCA
between the two morphotypes along the CV axis (Figure 10). Clear shape discrimina-
tion was also found between D. thiesseana and D. schlickumi shells after a permutation
Discriminant Function Analysis (T-squared = 236.3713, p (parametric) = 0.8335, p (per-
mutation) < 0.0001). However, after a permutation test with 1,000 replicates, only 54.2%
of “unknown” individuals (leave-one-out cross-validation) were correctly classied. Shape
changes along the CV1 are shown in Figure 11 and portray the tendency for D. thiesseana
to be slimmer compared to D. schlickumi and also to have a larger, i.e. elongated, aperture.
Environmental parameters
e Monte Carlo test in RDA analysis revealed two out of the six environmental pa-
rameters examined as signicantly dierent between the two species (p < 0.05): conduc-
tivity and Secchi depth. Depth and water temperature were excluded from the analysis
Figure 9. Principal component analysis of Procrustes coordinates for Dianella schlickumi (black circles)
and for Dianella thiesseana (open diamonds).
Rediscovery, discrimination and environmental correlates of Dianella species 49
Figure 10. Canonical Variate Analysis: Dark-grey bins stand for Dianella schlickumi specimens and light-
grey bins for Dianella thiesseana specimens.
Figure 11. Shape changes along the CV1: the shifts of landmark positions are indicated by straight lines.
Each line starts with a dot at the location of the landmark in the starting shape (i.e. Dianella schlickumi).
e length and direction of the line indicate the movement of the respective landmark towards the nal
shape (i.e. Dianella thiesseana).
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
50
due to their high ination factor (> 20). e rst and all canonical axes were statisti-
cally signicant (p = 0.044 and p = 0.002 respectively). e rst two ordination axes of
RDA explained 99.5% of the total species variance (Monte Carlo test, p < 0.05). Axis I
(eigenvalue 0.929, p < 0.05) was related to conductivity and DO (intra-set correlation
values 0.952 and -0.788 respectively) and Axis II (eigenvalue 0.066) to pH (intra-set
correlation value 0.428). D. schlickumi specimens from Lake Amvrakia were ordered at
the positive side of Axis I, having the highest conductivity values while D. thiesseana
specimens from the other lakes were ordered at the negative side of Axis I and correlated
with lower values of conductivity and higher values of DO (Figure 7, Table 2).
Discussion
Dianella schlickumi was recorded in the sublittoral zone of Lake Amvrakia in 1962
(Schütt 1962), but no exact details about this location were provided. e latest record
conrming the presence of the species in the lake was before 1983 (Radoman 1983).
After that, D. schlickumi was assumed to be extinct, as reported by Reischütz and Re-
ischütz (2002), Szarowska et al. (2005) and Albrecht et al. (2006), based on sampling
eorts in 1983, 2002 and 2003 respectively. Albrecht et al. (2011a) mentioned that
the originally recorded area of D. schlickumi is the northern extent of Lake Amvrakia.
At this part of the lake water, level uctuations were high during the last years, due to
insucient connectivity with the main body of the lake. In fact, during extremely dry
summers, this area is completely dry or water remains only in small pools. In such cases,
the predation pressure (mainly from sh) is intense, resulting in local extinction of mol-
luscs (Nilsson et al. 2008, Downing et al. 2010). Such rough conditions have probably
negatively aected the population size and the distribution of D. schlickumi in this lake.
Nevertheless, in our study, we were able to collect specimens of various ages at several
sampling sites, indicating that this snail is still extant and thriving in its type locality.
Under the IUCN Red List guidelines (IUCN 2017), D. schlickumi is considered as
“Critically Endangered (Possibly Extinct)” [following the criteria B1ab (i, iii)] (Albre-
cht et al. 2011a). However, since a viable population of this species was demonstrated
in the present study, the tag “Possibly Extinct” should be removed from its assessment.
e rediscovery of the species does not however improve its conservation status, as its
extent of occurrence is estimated to be less than 100 km2, restricted to the sublittoral
zone of Lake Amvrakia (<10 km2). Moreover, D. schlickumi is known to exist only in a
single location, where habitat degradation was also noticed due to agricultural practices
and pesticide contamination (omatou et al. 2013 a, b). Consequently, the classica-
tion of Critically Endangered should be maintained, as the IUCN criteria continue
to be met. As for D. thiesseana, it should also continue to be considered as Critically
Endangered [B1ab (ii, iii) and 2ab (ii, iii)] since it has an extent of occurrence of 94
km2 and an area of occupancy of 2 km2. Its habitat is also degraded because of intensive
agricultural practices, urban sewage, stock grazing land and small industries (Bertahas
et al. 2006).
Rediscovery, discrimination and environmental correlates of Dianella species 51
Both species thrive on soft substrate (Albrecht et al. 2011a, b). D. schlickumi seemed
to prefer slightly shallower habitats, from 3 m to 15 m, while D. thiesseana had a range
of distribution from 2.5 m to 27 m. e species absence from autumn surveys may be
due to depth migrations often observed in gastropods (Brown 2001). Even though this
study has conrmed the rediscovery of D. schlickumi, it does not clarify in detail the ac-
curate distribution of the species, which might be the subject of future research.
e anatomical characters of D. schlickumi known from literature (Schütt 1962) (i.e.
the radula) and those described in the present paper (i.e. the ctenidium and osphradium,
the central nervous system, the gastric caecum, the rectum and the male and female
reproductive organs) are similar to those reported by Radoman (1983) in the diagnosis
of the family Pyrgulidae Brusina 1881 (nominal subfamily Pyrgulinae) and of the genus
Dianella Gude, 1913 [(type species Dianella thiesseana (Kobelt, 1878)]. Similarities have
also been found between D. schlickumi and D. thiesseana in the nervous system, the gas-
tric caecum and the female reproductive organs as they are described for the latter species
by Szarowska et al. (2005, pp. 22–31 and related gures). However, a notable dierence
exists in the shape of the penis between the two species: the penis of D. thiesseana is wide,
with a triangular shape due to its expanded base and has a clear outgrowth on its left side
(Szarowska et al. 2005, p. 29, gs 32–37). e penis of D. schlickumi does not have an
expanded base and is gradually tapering without any distinct outgrowth.
D. schlickumi and D. thiesseana are discriminated due to their shell dimensions;
the rst one is smaller than the second (Radoman 1983). In our study, traditional and
geometric morphometric analyses support the assumption that D. thiesseana and D.
schlickumi are quite distinguishable in terms of external morphology. However, neither
in the original description of the two taxa (Kobelt 1878 and Schütt 1962 respectively)
nor in the subsequent studies (Radoman 1983, Szarowska et al. 2005), shell shape
appears to be signicantly dierent between the two taxa. Moreover, Szarowska et al.
(2005) reported that the shell characters and dimensions of D. schlickumi, described in
the literature and those of D. thiesseana from Lake Lysimachia, lie within the range ob-
served for D. thiesseana from Lake Trichonis. Even though, the intra-subspecic mor-
phometric variation is quite high, the two species are actually distinct in size and shape
(Figures 8–10). erefore, in the “Dianella” case, geometric morphometric analysis has
been proved to be a useful tool for exploring patterns of species diversity and seems
promising for examining morphological adaptations of these species. Specically, it is
clear that the Centroid Size, derived from Geometric Morphometric Analysis, can be
used safely to distinguish those two species.
In some cases, the environmental parameters set limits (Pyron and Brown 2015)
and may inuence (Økland 1983, Pip 1986, Falniowski 1987) the distribution of
freshwater snails. e analysis performed revealed conductivity, DO and pH as the
most signicant parameters explaining the current species variance. D. thiesseana, ac-
cording to RDA, seems to prefer more oxygenated waters with lower conductivity
values and pH than D. schlickumi. Conductivity, dissolved oxygen (e.g. Çabuk et al.
2004, Kazibwe et al. 2006, Cloherty and Rachlin 2011) and pH (Økland 1983, Pip
1986, Çabuk et al. 2004) are also referenced as key environmental proxies explaining
Chrysoula Ntislidou et al. / Nature Conservation 27: 35–58 (2018)
52
gastropods distribution in water bodies. Biological interactions and/or historical fac-
tors, which need further surveys and analyses, may also be responsible.
Both Dianella species require urgent conservation measures to be enforced and
suitable management plans to be implemented in the whole studied area, focusing
on the protection of these species and the improvement of their habitats. e studied
lakes form an ecologically important complex as Special Conservation areas (Council
Directive 92/43/EEC), listed in the NATURA 2000 network. However, the integrity
of local mollusc populations in these ecosystems is threatened due to the presence of
invasives; Ferrissia fragilis (Tryon, 1863) and Physa acuta Draparnaud, 1805 in lakes
Trichonis and Lysimachia (Albrecht et al. 2014) and of Potamopyrgus antipodarum
(J.E. Gray, 1843) in Lake Trichonis (Radea et al. 2008). e invasive impacts upon
native molluscs may be direct, through competition for food and space (Carlsson et al.
2004) or indirect, through changes in ecosystem function and/or parasitism (Brown
et al. 2008). e antagonistic behaviour of P. antipodarum and P. acuta is already well-
known (e.g. Schreiber et al. 2003, Zukowski and Walker 2009), although no such out-
comes have been documented for F. fragilis until now (Albrecht et al. 2014). Available
knowledge on the eects of invasive species on native mollusc species and especially on
D. thiesseana, is insucient to estimate the possible threats.
One of the main pressures identied in lakes Amvrakia, Trichonis and Lysimachia
is eutrophication from agriculture run-o and wastewaters (Bertahas et al. 2006,
omatou et al. 2013 a, b). Dianella inhabits the sublittoral zone of these lakes, which
is mainly aected by eutrophication (Pilotto et al. 2012). e development of water
quality indices based on zoobenthic communities and related to eutrophication will
help to assess the ecological status of these water bodies and prevent their further
deterioration. e impacts could be minimised by preserving natural vegetation or
establishing buer zones. Only low-impact activities should be allowed near the lakes
and harmful practices should be prohibited or regulated to a more distant zone. Fur-
thermore, it is crucial to restore the type locality of D. schlickumi by maintaining the
connectivity of the northern extension of Lake Amvrakia with the main lake water
body, thus preventing the species’ historical distribution area to become dry. Addi-
tional measures to reduce water abstraction could help the maintenance of the ecologi-
cal water level in the lakes, securing slight seasonal uctuations and preventing aquatic
biodiversity in general (Zohary and Ostrovsky 2011). Further temporal and spatial
surveys which will identify the ecological features, the ecosystem functions and the
habitat preferences of the studied species could signicantly support conservation ef-
forts. Environmental education and citizen awareness could additionally support con-
servation eorts for both endemic gastropods.
Conclusion
Our results conrm the rediscovery of the narrow endemic species Dianella schlic-
kumi in Lake Amvrakia since past sampling eorts did not detect it for more than
Rediscovery, discrimination and environmental correlates of Dianella species 53
30 years. Moreover, they provide basic knowledge on its anatomical, morphologi-
cal and distributional patterns and its discrimination from Dianella thiesseana. e
two species are discriminated by their shell size and shape. Moreover, the presence
of Dianella thiesseana is conrmed in lakes Trichonis and Lysimachia. We conclude
that further conservation measures have to be implemented for eective protection
of both species.
Acknowledgements
We would like to thank Mrs E Mavromati from the Greek Biotope/Wetland Centre
for providing us with data from the National Greek Monitoring Programme for the
presence of Dianella thiesseana in Lake Lysimachia.
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