Tree-based delimitation of morphologically ambiguous taxa: a study of the lizard malaria parasites on the Caribbean island of Hispaniola

Malaria parasites in the genus Plasmodium have been classified primarily on the basis of differences in morphology. These single-celled organisms often lack distinguishing morphological features, and this can encumber both species delimitation and identification. Six saurian malaria parasites have been described from the Caribbean island of Hispaniola. All six infect lizards in the genus Anolis, but only two of these parasites can be distinguished using morphology. The remaining four species overlap in morphology and geography, and cannot be consistently identified using traditional methods. We compared a morphological approach with a molecular phylogenetic approach for assessing the taxonomy of these parasites. We surveyed for blood parasites from 677 Anolis lizards, representing 26 Anolis spp. from a total of 52 sites across Hispaniola. Fifty-five of these lizards were infected with Plasmodium spp., representing several new host records, but only 24 of these infections could be matched to previously described species using traditional morphological criteria. We then estimated the phylogeny of these parasites using both mitochondrial (cytb and coxI) and nuclear (EF2) genes, and included carefully selected GenBank sequences to confirm identities for certain species. Our molecular results unambiguously corroborated our morphology-based species identifications for only the two species previously judged to be morphologically distinctive. The remaining infections fell into two well-supported and reciprocally monophyletic clades, which contained the morphological variation previously reported for all four of the morphologically ambiguous species. One of these clades was identified as Plasmodium floridense and the other as Plasmodium fairchildi hispaniolae. We elevate the latter to Plasmodium hispaniolae comb. nov. because it is polyphyletic with the mainland species Plasmodium fairchildifairchildi and we contribute additional morphological and molecular characters for future species delimitation. Our phylogenetic hypotheses indicate that two currently recognised taxa, Plasmodium minasense anolisi and Plasmodium tropiduri caribbense, are not valid on Hispaniola. These results illustrate that molecular data can improve taxonomic hypotheses in Plasmodium when reliable morphological characters are lacking.     

The oldest South American tuco-tuco (late Pliocene,northwestern Argentina) and the boundaries of the genus Ctenomys (Rodentia,Ctenomyidae)

A new species of Ctenomyidae from the late Pliocene of Uquía Formation (northwestern Argentina) is described. The new remains consist of a fragmentary rostrum, and a left mandible with partial lower dentition. Its phylogenetic affinity and morphological specializations for tooth-digging support its assignation to the South American rodent genus Ctenomys. In this context, we highlight the importance of unique morphological specializations for the delimitation of genera within an intrafamilial clade in which similar adaptive strategies could have evolved more than once. The new materials are the oldest fossils for the genus (ca. 3.5 Ma), and their finding in the central Andes agrees with previous hypotheses about the possible area of origin of Ctenomys. They precede by about one million years the presence of Ctenomys chapalmalensis in the Pliocene of the Pampean region of central Argentina, the oldest record previously known for the genus. Nevertheless, the new species does not contribute key information about ancestral character states for the genus beyond those already known through C. chapalmalensis. The phylogenetic, adaptive and even chronological information supplied by these new materials would be linked to the differentiation of the genus rather than to its origin.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

Integrative taxonomy and preliminary assessment of species limits in the Liolaemus walkeri complex (Squamata,Liolaemidae) with descriptions of?three?new species from Peru

Species delimitation studies based on integrative taxonomic approaches have received considerable attention in the last few years, and have provided the strongest hypotheses of species boundaries. We used three lines of evidence (molecular, morphological, and niche envelopes) to test for species boundaries in Peruvian populations of the Liolaemus walkeri complex. Our results show that different lines of evidence and analyses are congruent in different combinations, for unambiguous delimitation of three lineages that were “hidden” within known species, and now deserve species status. Our phylogenetic analysis shows that L. walkeri, L. tacnae and the three new species are strongly separated from other species assigned to the alticolor-bibronii group. Few conventional morphological characters distinguish the new species from closely related taxa and this highlights the need to integrate other sources of data to erect strong hypothesis of species limits. A taxonomic key for known Peruvian species of the subgenus Lioalemus is provided.     

Phylogeny, diversity, and species delimitation of the North American Round-Nosed Minnows (Teleostei: Dionda), as inferred from mitochondrial and nuclear DNA sequences

Accurate delimitation of species is a critical first step in protecting biodiversity. Detection of distinct species is especially important for groups of organisms that inhabit sensitive environments subject to recent degradation, such as creeks, springs, and rivers in arid or semi-desert regions. The genus Dionda currently includes six recognized and described species of minnows that live in clear springs and spring-fed creeks of Texas, New Mexico (USA), and northern Mexico, but the boundaries, delimitation, and characterization of species in this genus have not been examined rigorously. The habitats of some of the species in this genus are rapidly deteriorating, and many local populations of Dionda have been extirpated. Considering the increasing concerns over degradation of their habitat, and pending a more detailed morphological revision of the genus, we undertook a molecular survey based on four DNA regions to examine variation over the range of the genus, test species boundaries, and infer phylogenetic relationships within Dionda. Based on analyses of two mitochondrial (cytb and D-loop) and two nuclear (Rag1 and S7) DNA regions from specimens collected throughout the range of Dionda, we identified 12 distinct species in the genus. Formerly synonymized names are available for two of these species, and four other species remain undescribed. We also redefine the known range of six species. The limited distribution of several of the species, coupled with widespread habitat degradation, suggests that many of the species in this genus should be targets for conservation and recovery efforts.     

Using multilocus approach to uncover cryptic diversity within Pseudotrapelus lizards from Saudi Arabia

The agamid Pseudotrapelus lizards inhabit the mountainous areas of the Arabian Peninsula and eastern North Africa. Currently six Pseudotrapelus species are recognised, though diagnostic morphological characters are still lacking, creating great difficulty in describing new species. Recently, two specimens of Pseudotrapelus were collected from the vicinity of Riyadh in central Saudi Arabia, an area that was not sampled in previous phylogenetic studies. In here we used both mitochondrial and nuclear data to investigate the phylogenetic position of the new samples, and assess their phylogenetic relationships with the other recognised species of Pseudotrapelus from across the distribution range of the genus. We used a multilocus approach of haplotype networks, concatenated datasets and species trees, performed mitochondrial and nuclear species delimitation analyses, and estimated divergence times. In general, our results support previous molecular studies and uncover the presence of cryptic diversity within Pseudotrapelus. The phylogenetic structure of the genus is of two major clades and within them seven distinct, delimited phylogenetic groups belonging to the six recognised species and the seventh to the individuals from Riyadh. The Riyadh specimens were distinct in all analyses performed. We suggest that the new specimens from the Riyadh area are a distinct lineage, forming a clade with their phylogenetic relatives, P. sinaitus and P. chlodnickii. The clade formed by these three species diverged during the Late Miocene around 6.4 Ma, with cladogenesis possibly facilitated by vicariance and isolation caused due to climatic fluctuations and the progression of sandy areas. Our results suggest further morphological research is necessary to revise the taxonomic status of this lineage and of the entire genus.     

Compilation of morphological and molecular data,a necessity for taxonomy: The case of Hormogaster abbatissae sp. n. (Annelida,Clitellata, Hormogastridae)

Conflict among data sources can be frequent in evolutionary biology, especially in cases where one character set poses limitations to resolution. Earthworm taxonomy, for example, remains a challenge because of the limited number of morphological characters taxonomically valuable. An explanation to this may be morphological convergence due to adaptation to a homogeneous habitat, resulting in high degrees of homoplasy. This sometimes impedes clear morphological diagnosis of species. Combination of morphology with molecular techniques has recently aided taxonomy in many groups difficult to delimit morphologically. Here we apply an integrative approach by combining morphological and molecular data, including also some ecological features, to describe a new earthworm species in the family Hormogastridae, Hormogaster abbatissae sp. n., collected in Sant Joan de les Abadesses (Girona, Spain). Its anatomical and morphological characters are discussed in relation to the most similar Hormogastridae species, which are not the closest species in a phylogenetic analysis of molecular data. Species delimitation using the GMYC method and genetic divergences with the closest species are also considered. The information supplied by the morphological and molecular sources is contradictory, and thus we discuss issues with species delimitation in other similar situations. Decisions should be based on a profound knowledge of the morphology of the studied group but results from molecular analyses should also be considered.     

Rolling into the deep of the land planarian genus <Emphasis Type="Italic">Choeradoplana</Emphasis> (Tricladida,Continenticola, Geoplanidae) taxonomy

The land planarian genus Choeradoplana (Plathelminthes, Tricladida) is currently integrated by 13 species. In previous works, morphological variation in its type species, Choeradoplana iheringi, was reported, but no attempt to test whether it is just a single species has been made yet. In order to disentangle the taxonomy of this species and further members of the genus, we sampled new specimens and combined morphological and molecular data and also have evaluated the performance of diverse methods of molecular species delimitation. Our data point to the presence of two cryptic species named C. iheringi, plus two new species, all hidden under the same general appearance. An in-depth morphological study of the specimens allowed detection of diagnostic morphological traits in each species, for which we also propose a molecular diagnosis. This integrative taxonomic study demonstrates once again the usefulness of molecular tools to weigh minor morphological characteristics and thus reveal the existence of species that would otherwise remain cryptic. However, under certain parameters, the molecular methods may over-split species with a high genetic structure, maybe pointing to incipient speciation. This makes critical the use of these methods combined with a comprehensive morphological approach. We also present a comprehensive phylogenetic tree including most Choeradoplana species. The tree, well supported, allows making some preliminary inferences on the evolution of the group and its historical biogeography.     

Re-Establishment of the Genus Ania Lindl. (Orchidaceae)

Ania Lindl. is a small genus of the tribe Collabieae subtribe Collabiinae (Orchidaceae). For the last 150 years, it has generally been treated as a synonym of Tainia Blume. In this study, we critically re-examined morphological characters that have been used to distinguish Ania from Tainia, and assessed the phylogeny of Tainia using morphological and palynological characters. Sequences of the nuclear ribosomal ITS, chloroplast trnL intron and combined DNA data sets were analysed to clarify the delimitation and the phylogeny of these groups. The morphological and palynological survey revealed a number of useful diagnostic characters which permit a clear definition of Ania, after the exclusion of a single taxonomically questionable species. Results confirmed that Ania is distinct from Tainia. Phylogenetic reconstructions based on molecular data provided the greatest resolution and produced a morphologically well differentiated clade of Ania. In addition to morphological and suggested palynological characters, the phylogenies were also supported by karyological evidence. Our results support the independent generic status of Ania. The genus name Ania is revived and re-established.     

Testing Phylogenetic Hypotheses of the Subgenera of the Freshwater Crayfish Genus Cambarus (Decapoda: Cambaridae)

Background

The genus Cambarus is one of three most species rich crayfish genera in the Northern Hemisphere. The genus has its center of diversity in the Southern Appalachians of the United States and has been divided into 12 subgenera. Using Cambarus we test the correspondence of subgeneric designations based on morphology used in traditional crayfish taxonomy to the underlying evolutionary history for these crayfish. We further test for significant correlation and explanatory power of geographic distance, taxonomic model, and a habitat model to estimated phylogenetic distance with multiple variable regression.

Methodology/Principal Findings

We use three mitochondrial and one nuclear gene regions to estimate the phylogenetic relationships for species within the genus Cambarus and test evolutionary hypotheses of relationships and associated morphological and biogeographical hypotheses. Our resulting phylogeny indicates that the genus Cambarus is polyphyletic, however we fail to reject the monophyly of Cambarus with a topology test. The majority of the Cambarus subgenera are rejected as monophyletic, suggesting the morphological characters used to define those taxa are subject to convergent evolution. While we found incongruence between taxonomy and estimated phylogenetic relationships, a multiple model regression analysis indicates that taxonomy had more explanatory power of genetic relationships than either habitat or geographic distance.

Conclusions

We find convergent evolution has impacted the morphological features used to delimit Cambarus subgenera. Studies of the crayfish genus Orconectes have shown gonopod morphology used to delimit subgenera is also affected by convergent evolution. This suggests that morphological diagnoses based on traditional crayfish taxonomy might be confounded by convergent evolution across the cambarids and has little utility in diagnosing relationships or defining natural groups. We further suggest that convergent morphological evolution appears to be a common occurrence in invertebrates suggesting the need for careful phylogenetically based interpretations of morphological evolution in invertebrate systematics.     

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.     

Integrative taxonomy of the stunt nematodes of the genera Bitylenchus and Tylenchorhynchus (Nematoda,Telotylenchidae) with description of two new species and a molecular phylogeny

Stunt nematodes are characterized by phenotypic plasticity, with overlapping morphology and morphometry leading to potential misidentification. Consequently, the application of integrative taxonomic approaches is useful to species delimitation based on a combination of different perspectives, e.g. morphology and DNA sequences. We conducted nematode surveys in cultivated and natural environments in Spain and the USA, from which we identified 18 known species of the family Telotylenchidae and two new taxa within the studied samples. These species were morphologically, morphometrically, and molecularly characterized. The results of light and scanning electron microscopic observations, and molecular and phylogenetic analysis also allowed two new species to be distinguished, described herein as B itylenchus hispaniensis sp. nov. and T ylenchorhynchus mediterraneus sp. nov. The phylogenetic analysis was carried out using molecular data from nuclear ribosomal DNA genes [D2–D3 expansion segments of the large ribosomal subunit (28S), internal transcribed spacer (ITS), and partial small ribosomal subunit (18S)]. We also provide here a test of alternative hypotheses that confirms the monophyly of both Tylenchorhynchus and Bitylenchus sensu Siddiqi's classification but does not support Fortuner & Luc's conceptual view of Tylenchorhynchus as a large genus. Ancestral state reconstructions of several diagnostic morphological characters using a maximum parsimony approach showed congruence in morphological and molecular evolution for stylet knob inclination and tail tip annulation. Our analysis emphasizes some of the problems related to the taxonomy and phylogeny of nematodes of Telotylenchinae. © 2014 The Linnean Society of London     

Using 18S rDNA to resolve diaptomid copepod (Copepoda: Calanoida: Diaptomidae) phylogeny: an example with the North American genera

The phylogenetic relationships among the numerous genera of diaptomid copepods remain elusive due to difficulties in obtaining sufficient numbers of phylogenetically informative morphological characters for cladistic analysis. Molecular phylogenetic techniques offer high potential to resolve phylogenetic relationships in the absence of sufficient morphological characters because of the ease in which many characters can be unambiguously coded. I present the first molecular phylogeny for diaptomid copepod genera using 18S rDNA. Specifically, I test Light’s (1939) hypothesis regarding the interrelationships among the North American diaptomid genera. The 18S phylogeny is remarkably consistent with Light’s hypothesis. The endemic North American genera represent a monophyletic group exclusive of the non-endemic genera. Moreover, his hypothesized basal genus for the North America genera, Hesperodiaptomus, is the basal genus in this analysis. However, his Leptodiaptomus group is not reciprocally monophyletic with his Hesperodiaptomus group, but is rather a derived member of the latter group. Finally, the genus Mastigodiaptomus is found to be more closely allied with the non-endemic genera, as Light suggested. This phylogeny contributes heavily to the understanding of phylogenetic relationships among North American diaptomids and has large implications for the systematics of diaptomids in general. The use of 18S rDNA sequences in phylogenetic analyses of diaptomid copepods can be used to confirm the monophyly of recognized genera, the interrelationships among genera, and subsequent biogeographic interpretation of the family’s diversification. The use of molecular data, such as 18S rDNA sequences, to test phylogenetic hypotheses based on a very limited number of morphological characters will be a particularly useful approach to phylogenetic analysis in this system.     

Taxonomic implication of embryo micromorphology in the genus <Emphasis Type="Italic">Vicia</Emphasis> L. (Fabaceae)

In this work, embryos of selected Vicia species were examined to reveal the micromorphological characters that could have taxonomic significance. Experimental results show that morphological characters such as cotyledons shape and colour, radicle shape, colour and position, plumule colour and stipular parts attachments are variable and could be used for taxa identification in the genus Vicia. Numerical analysis based on 38 micromorphological characters was used to build a phenogram that indicated the relationships among the studied taxa. In addition, an identification key using embryo characters was prepared for Vicia species. The variability of embryo characters were discussed in the taxonomic context. Although the embryo characters have only limited phylogenetic signal for Vicia species, they might be used for their morphological delimitation.     

Phylogeny of the Atlantic and Pacific species of Ligophorus (Monogenea: Dactylogyridae): Morphology vs. molecules

Within ectoparasitic fish monogeneans, the genus Ligophorus contains a high number of species from which several were recently described. The precise determination of their taxonomic status requires robust diagnostic morphologic features that rely predominantly on a restricted set of sclerotized structures. In the present study, these morphological characters were used for the reconstruction of a phylogenetic tree, which was compared with a tree built from molecular data (28S and ITS1 DNA sequences). Thirty-eight morphological characters were used in 29 species of Ligophorus from the Atlantic and Pacific regions and 5 species within close genera of Dactylogyridae. The morphological and molecular phylogenetic trees are congruent and suggest that the genus Ligophorus is monophyletic, and that species parasitizing Liza spp. and Chelon labrosus occupy basal positions. The present study suggests that host switching is a common event in this host–parasite association, because about half of the species infecting the same host species are not close relatives. Following host switching, dispersal with vicariance is probably an important force shaping the present distribution and diversity of Ligophorus. The pattern of occurrence of Ligophorus spp. on Mugil cephalus supports that reproductive isolation and therefore parallel speciation are taking place among these parasitic organisms.