scholarly journals DNA barcodes combined with geometric morphometry challenge species hypothesis in palaemonid shrimp

2021 ◽  
Vol 4 ◽  
Author(s):  
Aleksandra Jablonska ◽  
Nicolas Navarro ◽  
Remi Laffont ◽  
Remi Wattier ◽  
Vladimir Pesic ◽  
...  
Keyword(s):  
Taxonomic Status ◽  
Balkan Peninsula ◽  
Integrative Approach ◽  
Shape Variation ◽  
Separate Species ◽  
Geometric Morphometric ◽  
The Balkans ◽  
Operational Taxonomic Units ◽  
Freshwater Shrimps ◽  
Coi Sequences

Although the Mediterranean Region is known as a hotspot for biodiversity and endemism its freshwater fauna is still greatly unexplored, and even the emblematic taxa such as decapods require in-depth integrative investigation. In our research we used integrative approach composed of various geometric morphometric and molecular methods to challenge the taxonomic status of two freshwater shrimps representing Palaemonidae: Palaemon antennarius and Palaemon minos. Basing on 352 COI sequences, three Molecular Operational Taxonomic Units (MOTUs) were defined. Two of them belonged to P. antennarius: first inhabiting Apennine Peninsula and Sicily, the second one from the Balkan Peninsula. The third MOTU corresponded to Palaemon minos from Crete. The Balkan MOTU of P. antennarius was closer to P. minos in terms of genetics, than to the other conspecific MOTU. The carapace shape variation, studied on 180 individuals, was mainly explained by the geographic distribution. Balkan and Cretan groups were clearly distinguished, while other samples were distributed along the shape gradient from Sicily and southern Apennine Peninsula to the Balkans. The results of our study showed that, either the MOTU assigned to the Apennine Peninsula and Sicily constitutes a separate species or, alternatively, P. minos should be synonymised with P. antennarius.

The Cheilosia canicularis group (Diptera: Syrphidae): species delimitation and evolutionary relationships based on wing geometric morphometrics

Zootaxa ◽  
2008 ◽  
Vol 1825 (1) ◽  
pp. 40 ◽  
Author(s):  
JASMINA LUDOŠKI ◽  
LJUBINKA FRANCUSKI ◽  
ANTE VUJIĆ ◽  
VESNA MILANKOV
Keyword(s):  
Sequence Data ◽  
Balkan Peninsula ◽  
Wing Shape ◽  
Canonical Variate ◽  
Wing Size ◽  
Shape Variation ◽  
Geometric Morphometric ◽  
Mahalanobis Distances ◽  
Size And Shape ◽  
Upgma Cluster Analysis

A landmark-based geometric morphometric approach was used to assess differences in the size and shape of wing among/within three species of the Cheilosia canicularis group (Diptera: Syrphidae): C. canicularis, C. himantopus and C. orthotricha. Wing size and shape variation was observed from 25, 176 and 41 specimens of C. canicularis, C. himantopus and C. orthotricha, respectively, collected from six localities on the Balkan Peninsula. Significant differences in wing size were obtained among the analysed species and canonical variate analysis showed that wing shape was sufficiently different to allow the correct classification of 73% individuals of C. canicularis, 80% of C. orthotricha and 94% of C. himantopus, and clear delimitation of the species pairs C. canicularis/C. orthotricha and C. himantopus/C. orthotricha. In all analysed species, the consistent sex dimorphism in wing shape was observed indicating that female specimens had shorter and broader wings than males. The UPGMA cluster analysis based on squared Mahalanobis distances revealed close accordance with previously published phylogenetic relationships of these species indicated by allozyme and DNA sequence data analysis. Our results suggested that wing parameters contain useful information in quantification phenotypic variation and identification of species in this challenging group for taxonomy and systematics.

A comprehensive and integrative re-description of Synchaeta tremula (Müller, 1786) and the newly rediscovered Synchaeta tremuloida Pourriot, 1965 (Rotifera: Synchaetidae)

Zootaxa ◽  
2017 ◽  
Vol 4276 (4) ◽  
pp. 503 ◽  
Author(s):  
TANJA WILKE ◽  
WILKO H. AHLRICHS ◽  
OLAF R.P. BININDA-EMONDS
Keyword(s):  
Taxonomic Status ◽  
Morphological Data ◽  
Integrative Approach ◽  
Similar Species ◽  
Ecological Data ◽  
Separate Species ◽  
Unique Character ◽  
Character Set ◽  
Initial Description ◽  
Scanning Electron

In this paper, we focus on the two morphologically similar species Synchaeta tremula (Müller, 1786) and Synchaeta tremuloida Pourriot, 1965. This study records one of the first detections of the latter species since its initial description and clarifies its uncertain taxonomic status. Using an integrative approach combining morphological data (from light and scanning electron microscopy) with molecular and ecological data, we present re-descriptions of S. tremula and S. tremuloida and confirm their status as separate species. Finally, we also provide a unique character set that can be readily applied to unambiguously identify each species using easily observable features. 

Disappearing apple snails (Caenogastropoda: Ampullariidae) of Thailand: a comprehensive update of their taxonomic status and distribution

2020 ◽  
Vol 86 (4) ◽  
pp. 290-305 ◽  
Author(s):  
Ting Hui Ng ◽  
Supunya Annate ◽  
Ekgachai Jeratthitikul ◽  
Chirasak Sutcharit ◽  
Yanin Limpanont ◽  
...  
Keyword(s):  
Cryptic Species ◽  
Taxonomic Status ◽  
Intraspecific Diversity ◽  
Human Consumption ◽  
Conservation Action ◽  
Separate Species ◽  
High Genetic Diversity ◽  
Geometric Morphometric ◽  
Geometric Morphometric Analysis ◽  
Species Taxonomy

ABSTRACT Ampullariidae include the largest of all freshwater snails and are of ecological, evolutionary and anthropogenic importance in Southeast Asia (SEA). Native ampullariids belonging to the genus Pila face various threats but are understudied, with their species taxonomy being confused and data on their distributions being scarce. We provide a comprehensive update on the nomenclature, status and distribution of Pila species in Thailand, based on DNA barcoding and geometric morphometric analysis of recently collected material. We confirm that at least five Pila species are extant in Thailand: Pila virescens, P. celebensis, P. turbinis, P. gracilis and P. pesmei. Pila celebensis, which has distinctive egg masses among all the known Southeast Asian Pila, appears to be sister to a clade comprising other SEA and some African Pila. Our results suggest that Pila may have dispersed into SEA on at least two separate occasions. Two singletons collected from northern and eastern Thailand may constitute separate species, but this requires further study. Intraspecific diversity of the mitochondrial cytochrome c oxidase subunit I gene was relatively high for P. celebensis, P. gracilis and P. pesmei (maximum uncorrected p-distances varied from 7% to 9%), and may include cryptic species. Conversely, P. virescens showed low intraspecific p-distances (c. 0%) among clades collected from different localities. This strongly suggests that introductions by humans may be the major cause of this pattern, and our own observations—we found that this species is being cultured and is commonly sold in markets for human consumption—are consistent with this. Pila turbinis was the rarest species, with live snails being collected from only two localities. Throughout Thailand, invasive confamilial Pomacea species appear to be replacing native Pila species, particularly in the Chao Phraya basin. While Thai Pila exhibit surprisingly high genetic diversity, with cryptic species likely being present, widespread invasive snails pose a major threat to their survival and urgent conservation action is needed.

Molecular phylogeny and species delimitation of the genus Dicerapanorpa (Mecoptera: Panorpidae)

2019 ◽  
Vol 187 (4) ◽  
pp. 1173-1195
Author(s):  
Gui-Lin Hu ◽  
Kai Gao ◽  
Ji-Shen Wang ◽  
Paul D N Hebert ◽  
Bao-Zhen Hua
Keyword(s):  
Species Delimitation ◽  
Phylogenetic Analyses ◽  
Integrative Approach ◽  
Geometric Morphometric ◽  
Operational Taxonomic Units ◽  
Integrative Analyses ◽  
Molecular Phylogenetic ◽  
Three New Species ◽  
Taxonomic Studies ◽  
Small Genus

Abstract Given that species is the fundamental unit in systematic biology, rigorous species delimitation is crucial for taxonomic studies, yet routine species delimitation remains an ongoing challenge in the taxonomic practice of insects. The two-horned scorpionfly Dicerapanorpa is a small genus in Panorpidae (Mecoptera) endemic to the Qinling-Bashan and Hengduan mountains, a biodiversity hotspot. However, species of Dicerapanorpa are difficult to delineate owing to marked intraspecific variation and interspecific similarity. Here, we investigate the diversity and species boundaries of Dicerapanorpa using an integrative approach based on DNA barcoding, morphological, geometric morphometric and molecular phylogenetic analyses. This integrative analyses confirmed the 13 described species of Dicerapanorpa and revealed three new species: Dicerapanorpa lativalva sp. nov., Dicerapanorpa hualongshana sp. nov. and Dicerapanorpa minshana sp. nov. Most molecular operational taxonomic units are in congruence with morphological clusters. Possible reasons for several discordances in Dicerapanorpa are tentatively discussed.

Morphological contrasts between Hedysarum spinosissimum ssp. spinosissimum and ssp. capitatum warrant recognition of separate species

1995 ◽  
Vol 73 (4) ◽  
pp. 566-572
Author(s):  
H. Baatout
Keyword(s):  
Mediterranean Basin ◽  
Taxonomic Status ◽  
Western Mediterranean ◽  
Point Of View ◽  
Stepwise Discriminant Analysis ◽  
Separate Species ◽  
Operational Taxonomic Units ◽  
Western Mediterranean Basin ◽  
Systematic Point ◽  
Floral Characters

The taxonomic status of the species complex Hedysarum spinosissimum L. (s.l.) has been evaluated on the basis of the phenetic variation within and between populations. Twenty-five characters were measured on plants grown from seeds of 9 populations of H. spinosissimum ssp. capitatum and 12 populations of H. spinosissimum ssp. spinosissimum from the western Mediterranean basin. Data were analyzed by means of multivariate methods. Stepwise discriminant analysis led to conclusions similar to those derived from principal components analysis. The results are discussed from a systematic point of view. On the basis of vegetative and floral characters, numerical analyses supported two morphologically distinct groups of operational taxonomic units corresponding to the predominantly selfing H. spinosissimum and the mostly outcrossing H. capitatum. Key words: Hedysarum spinosissimum L., phenetic variation, morphological contrast, Mediterranean basin, multivariate analysis.

scholarly journals Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod

2017 ◽  
Author(s):  
Michał Grabowski ◽  
Tomasz Mamos ◽  
Karolina Bącela-Spychalska ◽  
Tomasz Rewicz ◽  
Remi A Wattier
Keyword(s):  
Evolutionary History ◽  
28S Rdna ◽  
Cryptic Diversity ◽  
The Balkans ◽  
Operational Taxonomic Units ◽  
Freshwater Biota ◽  
History Of ◽  
Riverine Habitats ◽  
Coi Sequences ◽  
High Level

Background. The Balkans are a major biodiversity and endemism hotspot, worldwide. Among the freshwater biota, amphipods are known for their high cryptic diversity. However, little is known about the temporal and paleogeographic aspects of their evolutionary history. We used paleogeography as a framework for understanding the onset of diversification in Gammarus roeselii: 1) we hypothesised that, given the high number of isolated waterbodies in the Balkans, the species is characterised by high level of cryptic diversity, even on a local scale; 2) the long geological history of the region might promote pre-Pleistocene divergence between lineages; 3) given that G. roeselii thrives both in lakes and rivers, its evolutionary history could be linked to the Balkan Neogene paleolake system; 4) we inspected whether the Pleistocene decline of hydrological networks could have any impact on the diversification of G. roeselii. Material and Methods. DNA was extracted from 136 individuals collected from 26 sites all over Balkans. All individuals were amplified for ca. 650 bp long fragment of the mtDNA cytochrome oxidase subunit I (COI). After defining molecular operational taxonomic units (MOTU) based on COI, 50 individuals were amplified for ca. 900 bp long fragment of the nuclear 28S rDNA. Molecular diversity, divergence, differentiation and historical demography based on COI sequences were estimated for each MOTU. The relative frequency, geographic distribution and molecular divergence between COI haplotypes were presented as a median-joining network. COI was used also to reconstruct time-calibrated phylogeny with Bayesian inference. Probabilities of ancestors' occurrence in riverine or lacustrine habitats, as well their possible geographic locations, were estimated with the Bayesian method. A Neighbour Joining tree was constructed to illustrate the phylogenetic relationships between 28S rDNA haplotypes. Results. We revealed that G. roeselii includes at least 13 cryptic species or molecular operational taxonomic units (MOTUs), mostly of Miocene origin. A substantial Pleistocene diversification within-MOTUs was observed in several cases. We evidenced secondary contacts between very divergent MOTUs and introgression of nDNA. The Miocene ancestors could live in either lacustrine or riverine habitats yet their presumed geographic localisations overlapped with those of the Neogene lakes. Several extant riverine populations had Pleistocene lacustrine ancestors. Discussion. Neogene divergence of lineages resulting in substantial cryptic diversity may be a common phenomenon in extant freshwater benthic crustaceans occupying areas that were not glaciated during the Pleistocene. Evolution of G. roeselii could be associated with gradual deterioration of the paleolakes. The within-MOTU diversification might be driven by fragmentation of river systems during the Pleistocene. Extant ancient lakes could serve as local microrefugia during that time.

scholarly journals Geometric morphometric analysis of the skull of Podocnemis spp. (Pleurodira, Podocnemididae) and its implications on the taxonomic status of Podocnemis bassleri Williams, 1956

2015 ◽  
Author(s):  
Pedro S. R. Romano ◽  
Sergio A. K. Azevedo ◽  
Alexander W. A. Kellner ◽  
Gustavo R. Oliveira
Keyword(s):  
Multivariate Regression ◽  
Phylogenetic Signal ◽  
Ventral View ◽  
Taxonomic Status ◽  
Shape Variation ◽  
Diagnostic Features ◽  
Geometric Morphometric ◽  
Geometric Morphometric Analysis ◽  
Independent Variable ◽  
The Individual

Background. Podocnemis bassleri Williams, 1956 is the oldest definite record for Podocnemis, dating back to the Miocene of South America. However, the diagnosis of P. ba. is fragile, as assumed by Williams himself. Here we evaluated the hypothesis of P. ba. as a synonym of P. expansa by exploring the shape variation of the skull among Podocnemis spp. Methods. We collected 19 landmarks on the left side of the skull in ventral view from 33 Podocnemis spp. skulls. We included in the sample a new specimen of P. ba. from the Miocene of Acre, Brazil (MCT/RJ unnumbered) and the holotype (AMNH 1662) from the Miocene of Peru, along with: P. erythrocephala (n=3), P. ex. (n=9), P. lewyana (n=1), P. sextuberculata (n=9), P. unifilis (n=6), and P. vogli (n=3). The data was then Procrustes superimposed and a Relative Warps Analysis (RW) was performed to evaluate shape variation. Also, a multivariate regression of the Partial Warps (i.e.: the eigenvectors of the “bending energy” matrix) versus the log-transformed centroid size (the independent variable) was computed in order to evaluate the allometry in the data. All analyses were fulfilled using Rohlf’s TPS softwares. Results. The two first RWs accumulated ~52% of shape variation. The individual projection of the specimens in RW1 and RW2 showed a structure partially explained by a phylogenetic signal. P. ex. and P. se., previously considered as sister-groups, were projected in the negative scores of RW1. Also, these two species were projected, respectively, in the positive and negative scores of the RW2. The two specimens of P. ba. were nested among P. ex. specimens. Furthermore, the multivariate regression revealed an effect of the size in the shape variation: larger skulls have wider maxillae and posterior region laterally extended. Discussion. According to the literature, P. ex. and P. ba. are nearly indistinguishable (besides the larger size of the second), and Williams seems to have used the Miocene age of the second as a criterion for differentiating the two taxa. Indeed, one of the diagnostic features pointed out by Williams – slightly shorter relative distance from snout tip to posterior borders of the orbits – seems to be a taphonomic outcome. Moreover, the second diagnostic character – internal palatal processes of the maxillae abruptly dorsally projected – is extended anteroposteriorly in the MCT/RJ P. ba. specimen, as in all recent taxa. So, this character is polymorphic or also results of a taphonomic outcome. As consequence, P. ba. lacks a suitable diagnosis. Our preliminary results revealed little differences in shape of P. ex. and P. ba. and a clear allometric effect in the shape variation. In conclusion, it seems reasonable to suggest that P. ba. is a junior synonym of P. ex., although we will wait to claim it after additional analyses.

scholarly journals Geometric morphometric analysis of the skull of Podocnemis spp. (Pleurodira, Podocnemididae) and its implications on the taxonomic status of Podocnemis bassleri Williams, 1956

2015 ◽  
Author(s):  
Pedro S. R. Romano ◽  
Sergio A. K. Azevedo ◽  
Alexander W. A. Kellner ◽  
Gustavo R. Oliveira
Keyword(s):  
Multivariate Regression ◽  
Phylogenetic Signal ◽  
Ventral View ◽  
Taxonomic Status ◽  
Shape Variation ◽  
Diagnostic Features ◽  
Geometric Morphometric ◽  
Geometric Morphometric Analysis ◽  
Independent Variable ◽  
The Individual

Background. Podocnemis bassleri Williams, 1956 is the oldest definite record for Podocnemis, dating back to the Miocene of South America. However, the diagnosis of P. ba. is fragile, as assumed by Williams himself. Here we evaluated the hypothesis of P. ba. as a synonym of P. expansa by exploring the shape variation of the skull among Podocnemis spp. Methods. We collected 19 landmarks on the left side of the skull in ventral view from 33 Podocnemis spp. skulls. We included in the sample a new specimen of P. ba. from the Miocene of Acre, Brazil (MCT/RJ unnumbered) and the holotype (AMNH 1662) from the Miocene of Peru, along with: P. erythrocephala (n=3), P. ex. (n=9), P. lewyana (n=1), P. sextuberculata (n=9), P. unifilis (n=6), and P. vogli (n=3). The data was then Procrustes superimposed and a Relative Warps Analysis (RW) was performed to evaluate shape variation. Also, a multivariate regression of the Partial Warps (i.e.: the eigenvectors of the “bending energy” matrix) versus the log-transformed centroid size (the independent variable) was computed in order to evaluate the allometry in the data. All analyses were fulfilled using Rohlf’s TPS softwares. Results. The two first RWs accumulated ~52% of shape variation. The individual projection of the specimens in RW1 and RW2 showed a structure partially explained by a phylogenetic signal. P. ex. and P. se., previously considered as sister-groups, were projected in the negative scores of RW1. Also, these two species were projected, respectively, in the positive and negative scores of the RW2. The two specimens of P. ba. were nested among P. ex. specimens. Furthermore, the multivariate regression revealed an effect of the size in the shape variation: larger skulls have wider maxillae and posterior region laterally extended. Discussion. According to the literature, P. ex. and P. ba. are nearly indistinguishable (besides the larger size of the second), and Williams seems to have used the Miocene age of the second as a criterion for differentiating the two taxa. Indeed, one of the diagnostic features pointed out by Williams – slightly shorter relative distance from snout tip to posterior borders of the orbits – seems to be a taphonomic outcome. Moreover, the second diagnostic character – internal palatal processes of the maxillae abruptly dorsally projected – is extended anteroposteriorly in the MCT/RJ P. ba. specimen, as in all recent taxa. So, this character is polymorphic or also results of a taphonomic outcome. As consequence, P. ba. lacks a suitable diagnosis. Our preliminary results revealed little differences in shape of P. ex. and P. ba. and a clear allometric effect in the shape variation. In conclusion, it seems reasonable to suggest that P. ba. is a junior synonym of P. ex., although we will wait to claim it after additional analyses.

scholarly journals Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3016 ◽  
Author(s):  
Michał Grabowski ◽  
Tomasz Mamos ◽  
Karolina Bącela-Spychalska ◽  
Tomasz Rewicz ◽  
Remi A. Wattier
Keyword(s):  
Evolutionary History ◽  
28S Rdna ◽  
Cryptic Diversity ◽  
The Balkans ◽  
Operational Taxonomic Units ◽  
Freshwater Biota ◽  
History Of ◽  
Riverine Habitats ◽  
Coi Sequences ◽  
High Level

BackgroundThe Balkans are a major biodiversity and endemism hotspot, worldwide. Among the freshwater biota, amphipods are known for their high cryptic diversity. However, little is known about the temporal and paleogeographic aspects of their evolutionary history. We used paleogeography as a framework for understanding the onset of diversification inGammarus roeselii: (1) we hypothesised that, given the high number of isolated waterbodies in the Balkans, the species is characterised by high level of cryptic diversity, even on a local scale; (2) the long geological history of the region might promote pre-Pleistocene divergence between lineages; (3) given thatG. roeseliithrives both in lakes and rivers, its evolutionary history could be linked to the Balkan Neogene paleolake system; (4) we inspected whether the Pleistocene decline of hydrological networks could have any impact on the diversification ofG. roeselii.Material and MethodsDNA was extracted from 177 individuals collected from 26 sites all over Balkans. All individuals were amplified for ca. 650 bp long fragment of the mtDNA cytochrome oxidase subunit I (COI). After defining molecular operational taxonomic units (MOTU) based on COI, 50 individuals were amplified for ca. 900 bp long fragment of the nuclear 28S rDNA. Molecular diversity, divergence, differentiation and historical demography based on COI sequences were estimated for each MOTU. The relative frequency, geographic distribution and molecular divergence between COI haplotypes were presented as a median-joining network. COI was used also to reconstruct time-calibrated phylogeny with Bayesian inference. Probabilities of ancestors’ occurrence in riverine or lacustrine habitats, as well their possible geographic locations, were estimated with the Bayesian method. A Neighbour Joining tree was constructed to illustrate the phylogenetic relationships between 28S rDNA haplotypes.ResultsWe revealed thatG. roeseliiincludes at least 13 cryptic species or molecular operational taxonomic units (MOTUs), mostly of Miocene origin. A substantial Pleistocene diversification within-MOTUs was observed in several cases. We evidenced secondary contacts between very divergent MOTUs and introgression of nDNA. The Miocene ancestors could live in either lacustrine or riverine habitats yet their presumed geographic localisations overlapped with those of the Neogene lakes. Several extant riverine populations had Pleistocene lacustrine ancestors.DiscussionNeogene divergence of lineages resulting in substantial cryptic diversity may be a common phenomenon in extant freshwater benthic crustaceans occupying areas that were not glaciated during the Pleistocene. Evolution ofG. roeseliicould be associated with gradual deterioration of the paleolakes. The within-MOTU diversification might be driven by fragmentation of river systems during the Pleistocene. Extant ancient lakes could serve as local microrefugia during that time.

scholarly journals Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod

2017 ◽  
Author(s):  
Michał Grabowski ◽  
Tomasz Mamos ◽  
Karolina Bącela-Spychalska ◽  
Tomasz Rewicz ◽  
Remi A Wattier
Keyword(s):  
Evolutionary History ◽  
28S Rdna ◽  
Cryptic Diversity ◽  
The Balkans ◽  
Operational Taxonomic Units ◽  
Freshwater Biota ◽  
History Of ◽  
Riverine Habitats ◽  
Coi Sequences ◽  
High Level

Background. The Balkans are a major biodiversity and endemism hotspot, worldwide. Among the freshwater biota, amphipods are known for their high cryptic diversity. However, little is known about the temporal and paleogeographic aspects of their evolutionary history. We used paleogeography as a framework for understanding the onset of diversification in Gammarus roeselii: 1) we hypothesised that, given the high number of isolated waterbodies in the Balkans, the species is characterised by high level of cryptic diversity, even on a local scale; 2) the long geological history of the region might promote pre-Pleistocene divergence between lineages; 3) given that G. roeselii thrives both in lakes and rivers, its evolutionary history could be linked to the Balkan Neogene paleolake system; 4) we inspected whether the Pleistocene decline of hydrological networks could have any impact on the diversification of G. roeselii. Material and Methods. DNA was extracted from 136 individuals collected from 26 sites all over Balkans. All individuals were amplified for ca. 650 bp long fragment of the mtDNA cytochrome oxidase subunit I (COI). After defining molecular operational taxonomic units (MOTU) based on COI, 50 individuals were amplified for ca. 900 bp long fragment of the nuclear 28S rDNA. Molecular diversity, divergence, differentiation and historical demography based on COI sequences were estimated for each MOTU. The relative frequency, geographic distribution and molecular divergence between COI haplotypes were presented as a median-joining network. COI was used also to reconstruct time-calibrated phylogeny with Bayesian inference. Probabilities of ancestors' occurrence in riverine or lacustrine habitats, as well their possible geographic locations, were estimated with the Bayesian method. A Neighbour Joining tree was constructed to illustrate the phylogenetic relationships between 28S rDNA haplotypes. Results. We revealed that G. roeselii includes at least 13 cryptic species or molecular operational taxonomic units (MOTUs), mostly of Miocene origin. A substantial Pleistocene diversification within-MOTUs was observed in several cases. We evidenced secondary contacts between very divergent MOTUs and introgression of nDNA. The Miocene ancestors could live in either lacustrine or riverine habitats yet their presumed geographic localisations overlapped with those of the Neogene lakes. Several extant riverine populations had Pleistocene lacustrine ancestors. Discussion. Neogene divergence of lineages resulting in substantial cryptic diversity may be a common phenomenon in extant freshwater benthic crustaceans occupying areas that were not glaciated during the Pleistocene. Evolution of G. roeselii could be associated with gradual deterioration of the paleolakes. The within-MOTU diversification might be driven by fragmentation of river systems during the Pleistocene. Extant ancient lakes could serve as local microrefugia during that time.

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