Genetic variability and differentiation in Leuciscus (Telestes) souffia. Taxonomic and conservation inferences

Genetic variability of eight populations of Leuciscus (Telestes) souffia from France, Italy and Slovenia, and one of Leuciscus (Squalius) carolitertii from Spain was analysed using allozymic electrophoresis. For L (Telestes) souffia the parameters of variability ranged from P = 1 8.2 to 48.5 % and H_o = 0.023 to 0.092, values included in the global averages found for freshwater fishes. The phylogenetic relationships among these populations lead us to consider the souffia species belonging to the genus Telestes, and to subdivide it into two species: souffia (with at least two subspecies: souffia in France and agassizi in Slovenia), and muticellus. The genetic structure found within the French populations showed a certain differentiation between the Hérault Basin and the Var/Argens populations, recommending the preservation of these entities that could represent different adapted genetic pools. In the same way, the Italian region should be studied in detail, since the populations analysed from Bevera and Staffora Rivers (T. muticellus) showed evidence of a break of genetic flow (including a diagnostic locus between them).     

Tyrrhenian basins of Ligury as a new peri-Mediterranean ichthyogeographic district? Population structure of <Emphasis Type="Italic">Telestes muticellus</Emphasis> (Osteichthyes,Cyprinidae), a primary freshwater fish

The phylogeographic structure of vairone (Telestes muticellus), a primary freshwater fish endangered in a large part of its distributional range, was assessed: (i) to reconstruct the complex dispersion pattern in the upper Tyrrhenian hydrographic basins of Ligury, actually not recognised as peri-Mediterranean ichthyogeographic district, and (ii) to evidence the shape of population genetic structure as useful tool for future conservation strategies. A partial fragment of mitochondrial DNA cytochrome b (497 bp) was sequenced in 109 specimens sampled from eight populations, along an east–west geographic gradient. Fourteen haplotypes were identified, confirming the evolutionary distance between the two co-generic species: T. muticellus as ‘Ligurian’ clade and T. souffia as ‘French’ clade. The Nested Clade Analysis (NCA), the population genetic variability and population structure suggested a natural colonization occurred throughout the crossing of Alpine and Apennine watershed. The hierarchical Analysis of Molecular Variance (AMOVA) confirmed a geographic distinction between the populations from western (WTL) and from eastern (ETL) Tyrrhenian basins of Ligury colonised through the river capture processes along the Maritime Alpine watershed (Padano-Venetian district) and along the Apennine watershed (Tuscano-Latium district), respectively. Our results, evidencing the lack of genetic contiguity among vairone populations of the upper Tyrrhenian hydrographic basins of Ligury, allowed to recognise the presence of two management units (MUs) for its conservation. Handling editor: Christian Sturmbauer     

Microsatellites from the vairone Leuciscus souffia (Pisces: Cyprinidae) and their application to closely related species

Eleven microsatellites were isolated from the vairone Leuciscus souffia (Risso 1826), an endangered fish that inhabits river systems in and around the Alps in Europe. The level of genetic diversity was assessed in 29 individuals of the subspecies L. s. souffia, and their variability was further estimated in seven individuals of a different subspecies, L. s. muticellus. Eight of these microsatellite loci were also applied to seven closely related cyprinid species. Availability of the reported microsatellite loci will facilitate the investigation of population genetic structure of these species with applications for the development of conservation strategies and phylogeographical approaches.     

Testing Species Delimitations in Four Italian Sympatric Leuciscine Fishes in the Tiber River: A Combined Morphological and Molecular Approach

Leuciscine fishes represent an important component of freshwater ichthyofauna endemic to northern Mediterranean areas. This lineage shows high intra-specific morphological variability and exhibits high levels of hybridization, two characteristics that contribute to systematic uncertainties, misclassification of taxa and, potentially, the mismanagement of biodiversity. This study focused on brook chub, Squalius lucumonis, an endemic taxon of Central Italy. The taxonomic status of this species has long been questioned, and a hybrid origin from sympatric leusciscines (S. squalus x Rutilus rubilio, or S. squalus x Telestes muticellus) has been hypothesised. A phenotypic (evaluating shape and meristic counts) and genetic (using mitochondrial and nuclear markers) investigation of these four taxa was conducted to test species delimitation in sympatric areas and to evaluate the taxonomic status of S. lucumonis. One hundred and forty-five individuals of all four taxa were collected within streams of the lowest portion of the Tiber River basin and analysed; this region encompasses a large portion of the S. lucumonis distribution. The different morphological and genetic approaches were individually examined, compared, and then combined in a quantitative model to both investigate the limits of each approach and to identify cases of misclassification. The results obtained confirm the cladogenetic non-hybrid origin of S. lucumonis, highlight the need for immediate conservation actions and emphasise the value of an integrated approach in the study of leuciscines evolution.     

First report on the spawning migration and early life development of a cyprinid species of the genus Telestes

Reproductive migration of a Telestes species is reported for the first time for the Balkan endemic Telestes pleurobipunctatus (Stephanidis, 1939) from Louros River (Northwestern Greece), while the embryonic and larval development is also described for the first time for this species and genus. Adult fish migrate in late winter upstream and spawn in a peak of 2–3 nights in the outflow of springs. Embryos at hatching measure approx. 6.0 mm notochord length (NL), flexion starts at 8.1 mm NL and food particles are noted in the digestive tract of larvae at 8.2 mm NL. The larval period is characterized by the sequential formation of fins and the development of scalation. Fin differentiation ends with the completion of pelvic fin rays and the disappearance of the pre-anal finfold at 20.0 mm. First scales appear at 17.0 mm standard length (SL) and scalation completes at 27.8 mm SL. We propose that the size at a specific ontogenetic event, the pigmentation pattern and the pre-anal myomeres' number comprise diagnostic characters that permit distinguishing T. pleurobipunctatus larvae from other sympatric cyprinids. We note the rare pattern of scale scalation and pigmentation pattern of the target species. Parapatric speciation in T. pleurobipunctatus possibly related to its spawning migration pattern, as well as the use of larval identification in monitoring and conservation programmes targeting threatened cyprinids exhibiting this type of reproductive behaviour are also discussed.     

Phylogeography of the Italian vairone (<Emphasis Type="Italic">Telestes muticellus</Emphasis>, Bonaparte 1837) inferred by microsatellite markers: evolutionary history of a freshwater fish species with a restricted and fragmented distribution

Background  

Owing to its independence from the main Central European drainage systems, the Italian freshwater fauna is characterized by a high degree of endemicity. Three main ichthyogeographic districts have been proposed in Italy. Yet, the validity of these regions has not been confirmed by phylogenetic and population genetic analyses and a phylogeographic scenario for Italy's primary freshwater fish fauna is still lacking. Here, we investigate the phylogeography of the Italian vairone (Telestes muticellus).     

Phylogeography of the vairone (Leuciscus souffia,Risso 1826) in Central Europe

The vairone Leuciscus souffia is a cyprinid fish that inhabits river systems in and around the Alps. The complete mitochondrial DNA control region (945 bp) was sequenced in 295 vairone from 22 populations in Central Europe. A total of 51 haplotypes were identified with a maximum pairwise distance between haplotypes of 5.6%. Phylogenetic analyses revealed two major clades in L. souffia, an ‘Italian’ clade, and an ‘Alpine’ clade. Two hybrid zones exist, in the Mediterranean Alps and in the Soca basin. The position of the sister species of L. souffia, L. turskyi, to the ‘Alpine’ and the ‘Italian’ clade could not be resolved unambiguously. However, a linearized tree analysis indicated that L. turskyi represents a third lineage, that originated at the same time as the ‘Alpine’ and the ‘Italian’ clades of L. souffia. In the ‘Alpine’ clade two groups were resolved, a subclade with haplotypes from the Rhône and Var basins and a cluster with haplotypes from the Danube and Rhine systems. Our data suggest a long history of the vairone in Central Europe, predating Pleistocene glacial cycles. Two main refugia during glaciations must have existed, one in Italy and another one most probably in the Danube system. However, age estimates based on molecular clock calibrations suggest the survival of ‘Alpine’ haplotypes in several drainages during the last glaciation cycles. The Rhine system was only recently colonized.     

Mitochondrial capture and incomplete lineage sorting in the diversification of balitorine loaches (Cypriniformes,Balitoridae) revealed by mitochondrial and nuclear genes

Tang, Q.‐Y., Liu, S.‐Q., Yu, D., Liu, H.‐Z. & Danley, P.D. (2012) Mitochondrial capture and incomplete lineage sorting in the diversification of balitorine loaches (Cypriniformes, Balitoridae) revealed by mitochondrial and nuclear genes. —Zoologica Scripta, 41, 233–247. Understanding the diversification of species is a central goal of evolutionary biological studies. One powerful tool to investigate the speciation process is molecular systematics. Here, we use molecular methods to investigate the evolution of balitorine loaches belonging to two genera, Lepturichthys and Jinshaia. Both genera contain only two species (Lepturichthys fimbriata, Lepturichthys dolichopterus and Jinshaia sinensis and Jinshaia abbreviata), all of which are endemic to China. These species share many morphological and ecological characters and exhibit overlapping distributions in the Upper Yangtze River. In this study, we used two mitochondrial genes (Cytb and COI) and one nuclear gene (RAG1) to investigate the phylogenetic relationships within and between these two genera. Phylogenetic analyses and network construction based on mitochondrial and nuclear genes consistently supported the monophyly of Jinshaia. In contrast, the mitochondrial and nuclear genes yielded conflicting results in Lepturichthys. The phylogenetic analyses of mitochondrial sequences identify two distinct Lepturichthys lineages, Lepturichthys A and Lepturichthys B. Lepturichthys A includes most of L. fimbriata individuals from the Upper Yangtze River and is the sister group to all Jinshaia species. Lepturichthys B consists of the remaining L. fimbriata individuals from the Upper and Middle Yangtze River, and all L. dolichopterus individuals from the Minjiang River in Southeastern China. However, the analysis of the nuclear sequence indicates that the genus Lepturichthys is monophyletic and is only distantly related to Jinshaia. This incongruence suggests that introgressive hybridization might have occurred between L. fimbriata (Lepturichthys A) and Jinshaia species. As a result of this hybridization event, L. fimbriata captured the mitochondrial genome of the sympatric Jinshaia species. This capture event appears to have occurred at least 1.74 million years ago. Additionally, L. fimbriata appears to be paraphyletic; the nuclear data indicated that L. dolichopterus forms a monophyletic clade nested within L. fimbriata. Because L. dolichopterus and L. fimbriata are allopatric and hybridization may not be possible, we suggest that the observed paraphyly of L. fimbriata is a product of incomplete lineage sorting. In addition, the reciprocal monophyly of J. sinensis and J. abbreviata could not be resolved. This may be the result of interspecific hybridization as these species occur sympatrically. However, incomplete lineage sorting may have caused the observed topology of the Jinshaia species. The data presented here illustrate the complex evolutionary history of the balitorine loach species: intergeneric hybridization and interspecific hybridization have likely occurred in this lineage. In addition, possible incomplete lineage sorting may further obscure the evolutionary history of this group. The complex relationships of the balitorine loaches provide a rich evolutionary system to study the creation of sympatric and sister species.     

Biosystematics and evolutionary studies in Indian Drimia species

Abstract The taxonomic position and genetic relationship within Indian Drimia species is controversial due to their morphological similarities and genomic complexities. The present work gives an insight on the genetic relationship between Indian Drimia species on the basis of their karyotype, pollen morphology, flower opening characteristics, hybridization behavior, and by use of DNA sequence of two molecular markers (internal transcribed spacers [ITS] and maturase K [matK]). The karyotypic studies of Indian Drimia species revealed various polyploid forms making their identification and delimitation more difficult. The five species of Indian Drimia are grouped into two complexes, indica complex and wightii complex on the basis of their pollen morphology, karyotype, and hybridization behavior. These two groups were found to be evolving separately. The cytomorphological studies of wightii complex revealed that it is evolving through polyploid and chromosome repatterning, while indica complex have adapted polyploid as well as hybridization for evolution. Phylogeny obtained from DNA sequences of molecular markers (ITS and matK) confirmed that the indica complex and wightii complex are evolving parallely, by grouping them in two clusters. Thus, a combination of conventional and molecular methods proved to be of great use for delimiting a small but complex group of Indian Drimia species.     

Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes

Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA₃-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a “leuciscine” cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes.     

Molecular identification of <Emphasis Type="Italic">Schoenoplectiella</Emphasis> species (Cyperaceae) by use of microsatellite markers

Over the past few decades, use of molecular markers for species delimitation has drastically increased. Schoenoplectiella Lye has been recognized as a taxonomically difficult genus because of its morphological simplicity and frequent interspecific hybridization. The main objective of this study was to clarify the taxonomic identities of eight Schoenoplectiella species by use of molecular markers. We also evaluated the genetic relationships among S. × trapezoidea, known as a natural hybrid, and its close relatives. We used six microsatellite markers for 44 individuals from 31 natural populations of eight Schoenoplectiella species in South Korea. Six microsatellite marker combinations generated 59 amplification-detectable bands, of which 33 were specifically detected in one or more individuals of each species. Cluster analysis revealed that the grouping was consistent with the taxonomically recognized species. Our results do not support the hybrid origin of S. × trapezoidea. Rather, they suggest that this species is more closely related to S. hotarui. The informative microsatellite markers enabled us to clarify the distinctions among Schoenoplectiella species from South Korea and to identify the genetic relationships among these species. The molecular signatures found suitable for accurate identification of Schoenoplectiella species can be reliably used for studies of the phylogeny and evolution of this genus.     

Phylogenetic analyses of Tolpis Adans. (Asteraceae) reveal patterns of adaptive radiation,multiple colonization and interspecific hybridization

The plant genus Tolpis (Asteraceae) has been the subject of several investigations on the evolution of oceanic island plants. Its insular species were utilized in studies of artificial hybrid fertility, testing the validity of Baker’s law, the application of DNA barcodes, and the phylogenetic utility of inter‐simple sequence repeat markers. Despite this considerable interest in Tolpis, little is known about its phylogenetic history. Past investigations were unable to resolve most of the interspecific relationships, especially within the Canary Islands, where the genus is particularly diverse. Incomplete taxon sampling, the use of ambiguous outgroups and the limited utility of slowly evolving chloroplast DNA markers precluded detailed reconstructions. The present investigation presents a comprehensive molecular phylogeny of Tolpis. By utilizing highly variable nuclear DNA markers and a comprehensive taxon set, we have resolved the majority of interspecific relationships in the genus. Evaluations of competing tree topologies and ancestral area reconstructions complemented the analyses. Our results highlight the presence of three dominant mechanisms of island plant evolution—island colonization, adaptive radiation and interspecific hybridization—in Tolpis: (i) the extant distribution of the genus is the result of two independent colonization pathways, (ii) Tolpis has colonized at least one archipelago multiple times, (iii) the present insular diversity is the product of adaptive radiation, (iv) potential hybridization was detected between species now inhabiting different islands and archipelagoes, indicating sympatric historical distributions, and (v) several undescribed species await taxonomic recognition.     

Molecular and morphological patterns across Acanthocyclops vernalis‐robustus species complex (Copepoda,Cyclopoida)

Bláha, M., Hulák, M., Slouková, J. & Tě?itel, J. (2010). Molecular and morphological patterns across Acanthocyclops vernalis‐robustus species complex (Copepoda, Cyclopoida).—Zoologica Scripta, 39, 259–268. Morphological traits within Acanthocyclops (Kiefer, 1927) are highly variable, and morphology is too constrained to give complete information of phylogenetic relationships. This study combined morphological and molecular techniques to investigate the taxonomic and phylogenetic relationships of three species of Acanthocyclops (Acanthocyclops trajani, Acanthocyclops einslei and Acanthocyclops vernalis) inhabiting continental Europe. Morphological indices subjected to principal component analysis (PCA) separated sample populations into three distinct clusters corresponding with the taxonomic status of the species analysed. In addition, the taxonomy status of A. trajani and A. einslei was in agreement with molecular data; however, the intraspecific variation in sequences of 12S rRNA was lower in contrast to specimens morphologically determined as A. vernalis, which were divided into two deeply divergent clades, based on mtDNA sequence divergences. Moreover, high sequence divergence (26%) between these clades indicated the existence of another species that may not be a sister taxon of A. vernalis s.s. Results point to the need for further taxonomic work on Acanthocyclops.     

Primate numts and reticulate evolution of capped and golden leaf monkeys (Primates: Colobinae)

A recent phylogenetic study of langurs and leaf monkeys of South Asia suggested a reticulate evolution of capped and golden leaf monkeys through ancient hybridization between Semnopithecus and Trachypithecus. To test this hybridization scenario, I analysed nuclear copies of the mitochondrial cytochrome b gene (numts) from capped, golden and Phayre’s leaf monkeys. These numts were aligned with mitochondrial cytochrome b sequences of various species belonging to the genera Semnopithecus and Trachypithecus. In the phylogenetic tree derived from this alignment, the numts fell into three distinct clades (A, B and C) suggesting three independent integration events. Clade A was basal to Semnopithecus, and clades B and C were basal to Trachypithecus. Among the numts in clades A and C were sequences derived from species not represented in their respective sister mitochondrial groups. This unusual placement of certain numts is taken as additional support for the hybridization scenario. Based on the molecular dating of these integration events, hybridization is estimated to have occurred around 7.1 to 3.4 million years ago. Capped and golden leaf monkeys might have to be assigned to a new genus to reconcile their unique evolutionary history. Additionally, northeast India appears to be a ‘hot spot’ for lineages that might have evolved through reticulate evolution.     

Relationships among pansies (Viola section Melanium) investigated using ITS and ISSR markers

Sequences of the nuclear region ITS and the variable molecular markers ISSR were used to estimate the phylogeny of the section Melanium of the genus Viola. We confirm that the so-called pansies form a derived and monophyletic group. Two floral characters, the upturned side petals and the large size of pollen grains appear to be synapomorphies in Melanium. The Melanium species are very closely related, as shown by the reduced genetic variation compared to the other sections of Viola. Our analyses suggest x=5 or x=7 as the possible base chromosome number of the section Melanium. Polyploidy and hybridization would have played an important role in the evolutionary history of this clade resulting in a wide range of chromosome number. The low genetic differentiation and the complex cytological evolution suggest that diversification in Melanium is the result of a reticulate evolution and rapid radiation in Europe and Northern Africa.     

Interspecific hybridization in Rorippa (Brassicaceae): Patterns and processes

The current knowledge on natural hybridization between European lowland Rorippa species is reviewed. Morphological, cytological and molecular markers provide substantial evidence that four Rorippa species (R. amphibia, R. austriaca, R. palustris and R. sylvestris) are involved in hybridization processes. The main factors promoting initial hybridization events between Rorippa species in Europe are (1) natural and anthropogenic habitat disturbance, (2) the invasion of non‐native R. austriaca into western central Europe and (3) the self‐incompatibility system of R. amphibia, R. austriaca and R. sylvestris coupled with clonal growth. Outcomes of hybridization between European Rorippa species show a broad range from incidental hybrids through the formation of hybrid swarms to the evolution of new hybrid taxa. Rorippa × armoracioides is certainly the most successful Rorippa hybrid. It results from hybridization between R. austriaca and R. sylvestris and is known from many localities in the native range of R. austriaca and in regions invaded by R. austriaca. Within this system hybrid zones of different age provide the rare opportunity to analyse different stages of hybrid formation and hybrid speciation in natural populations.     

Conflicting mitochondrial and nuclear paraphyly in small‐sized West African house bats (Vespertilionidae)

Vallo, P., Benda, P., Červený, J. & Koubek, P. (2012). Conflicting mitochondrial and nuclear paraphyly in small‐sized West African house bats (Vespertilionidae). —Zoologica Scripta, 42, 1–12. Hybridization between species may result in introgression of mitochondrial DNA from one species to another. Phylogenetic inference, therefore, may not recover true evolutionary relationships. In bats, there are only a few reported cases of introgressive hybridization. House bats are a genus with obscure phylogeny and taxonomy, caused mainly by morphological similarity. We undertook a detailed analysis of small‐sized West African house bats (Scotophilus), tentatively identified as S. nigritellus, to clarify relationships between two sympatric colour forms. These forms were recovered in paraphyletic position to each other in both mitochondrial and nuclear phylogenies, signifying that they are two distinct species. While the yellow‐bellied form could be assigned beyond doubt to S. nigritellus s. str., the white‐bellied form may be an as yet undescribed species. Moreover, the white‐bellied form clustered as a sister mitochondrial lineage to another species, Scotophilus leucogaster. These sister lineages differed by only 2.6–2.8% sequence divergence, which lies within the intraspecific range for this genus. Two nuclear markers, however, contradicted the sister relationship, showing them instead to be distantly related. The apparent conflict between the mitochondrial and nuclear signals suggests that past hybridization may have occurred between these morphologically distinct species.     

Cryptic speciation in the Merodon luteomaculatus complex (Diptera: Syrphidae) from the eastern Mediterranean

The Merodon aureus group is characterized by high endemism and the presence of morphologically cryptic species. Within one of its subgroups, M. bessarabicus, seven species and four more species complexes have been described to date. One of these complexes, the M. luteomaculatus, comprises new taxa that are the subject of the present study. Its members have allopatric ranges restricted to the Balkan Peninsula and Aegean islands. This complex exhibits morphological variability that could not be characterized using a traditional morphological approach. Thus, we used integrative taxonomy with independent character sets (molecular, geometric morphometric, distributional) to delimit species boundaries. Data on three molecular markers (COI, 28S rRNA, and ISSR) and geometric morphometry of the wing and male genitalia, together with distributional data, enabled recognition of six cryptic species within the complex: M. andriotes sp. n., M. euri sp. n., M. erymanthius sp. n., M. luteomaculatus sp. n., M. naxius sp. n., and M. peloponnesius sp. n. We discuss the possible influence of Aegean paleogeographical history on the speciation of this complex.     

Homoplastic evolution of external colouration in Asian stout newts (Pachytriton) inferred from molecular phylogeny

Wu, Y., Wang, Y., Jiang, K., Chen, X. & Hanken, J. (2009). Homoplastic evolution of external colouration in Asian stout newts (Pachytriton) inferred from molecular phylogeny.—Zoologica Scripta, 39, 9–22. The Asian stout newts of the genus Pachytriton (Salamandridae) inhabit montane streams in south-eastern China. Despite their abundance in the pet trade, the phylogeny and systematics of this genus are poorly understood. Colouration is often used to delimit species under the assumption that consistent chromatic differences characterize independent evolutionary lineages. We present the first phylogenetic study of Pachytriton that incorporates 2.35 kb of mitochondrial DNA (ND2, cytb) and 1.2 kb of nuclear sequence data (RAG-1) along with morphometric characters to infer evolutionary relationships and patterns of colour evolution among the three described species: Pachytriton brevipes, Pachytriton labiatus and Pachytriton archospotus. Our results support the monophyly of Pachytriton and recover P. archospotus as the sister taxon to P. brevipes. Monophyly of P. labiatus is significantly rejected: south-western populations are sister to the group of P. brevipes plus P. archospotus, whereas north-eastern populations nest with P. brevipes. The two geographic units are further separated by multivariate morphological analyses. South-western P. labiatus is the type species; misidentification of north-eastern populations as P. labiatus results from their similar colouration. An unspotted, dark brown dorsum is the likely ancestral state for the genus, whereas black-spotted colouration characterized the common ancestor of P. brevipes, P. archospotus, and north-eastern P. labiatus and was secondarily lost in the latter group. Homoplastic evolution and intraspecific variation render colour pattern in Pachytriton an unreliable character for delimiting species boundaries. North-eastern populations of P. labiatus are declining as the result of human collection and habitat destruction and are in urgent need of conservation protection.