Patterns of cryptic hybridization revealed using an integrative approach: a case study on genets (Carnivora, Viverridae, Genetta spp.) from the southern African subregion

Recent years have seen the development of molecular‐based methodologies to investigate hybridization and its impact on the evolutionary process. However, morphological characterization of hybrid zones has only scantily been considered, especially in zootaxa. Thus, the level of congruence between molecular and morphological characters when attempting to detect hybrids remains a poorly tackled area. The genets (genus Genetta) provide an ideal case study for further investigation of the respective contribution of morphology and DNA in hybrid zone characterization because (1) their morphology has recently been exhaustively explored and (2) the existence of hybrid zones in southern Africa was proposed in the literature. We assessed levels of hybridization among the southern African genets, and questioned the role of ecological factors on the hybridization patterns detected. We used an integrative approach involving nine discrete morphological characters and a diagnostic discriminant function, geometric morphometrics and sequences of cytochrome b including collection specimens. The combination of independent materials allowed us to accurately reassess the level of hybridization in southern African genets, and revealed cryptic, interspecific gene flows. Morphology unambiguously detected a low number of G. maculata × G. tigrina hybrids and rejected the hypothesis of a large intergradation zone in KwaZulu‐Natal, thus supporting the species status of the two genets. Cytochrome b analyses revealed: (1) cryptic, massive hybridization between G. tigrina and the sympatric G. felina, and (2) a trace of reticulation (one sequence) between G. tigrina and the allopatric G. genetta. The type specimen of G. mossambica Matschie, 1902 is considered to be a morphological hybrid between G. maculata and G. angolensis. Remarkably, the morphological approaches (discrete characters and morphometrics) proved complementary to conclusions derived from cytochrome b sequences. Whilst morphometrics was generally unable to accurately identify all putative hybrids, this approach revealed diagnostic cranial shape differences between recognized species as well as the cryptic G. ‘letabae’ (included in the super‐species G. maculata). Morphometrics also confirmed the diagnostic value and age dependency of discrete characters. Our integrative approach appeared necessary to the detection of cryptic hybridizations and to the comprehensive characterization of hybrid zones. The recurrent detection of hybrids exhibiting tigrina‐like coat patterns may suggest (1) asymmetric hybridization of G. tigrina males to females of other species and (2) positive selection for tigrina‐like phenotype in South African habitats, but these hypotheses will have to be further tested using other sources of evidence. Despite the precise mosaic of hybrid zones identified in southern African genets, the environmental factors that shape patterns of distribution of hybrids remain unclear. Nevertheless, in the light of our range reassessment, it appears that seasonality of precipitation and periods of annual frost may play stringent roles in the distribution of genets. The complementarity of our results based on morphology and molecules is regarded as encouraging for the further development of integrative approaches in order to better understand the complex phenomena that underlie hybridization processes. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86 , 11–33.     

Genets and 'genet-like' taxa (Carnivora, Viverrinae): phylogenetic analysis, systematics and biogeographic implications

The subfamily Viverrinae is a taxon of uncertain systematic status. This study consists of cladistic analyses based on morphological characters of specimens belonging to the genera Genetta , Osbornictis , Poiana and Prionodon . Two levels of analysis are carried out, one concerning generic relationships (intergeneric analysis) and one dealing with the interrelationships of species within the genus Genetta (intrageneric analysis). In the first analysis, different outgroups were used in order to test the ingroup topology.
With regard to the intergeneric analysis, Osbornictis , Poiana and Prionodon , together with Genetta johnstoni , constitute a monophyletic group (including Nandinia ), which is the sister-group of a clade formed by the other species of genets. Thus, the genus Genetta is regarded as paraphyletic. Prionodon appears to be a derived taxon. The Poiana – Prionodon clade is well supported, especially by ultrastructural hair characters. The cladogram topology in the intrageneric analysis indicates an ecological transition from the rain forest genets to the savanna genets. This supports a rain forest origin of the genus Genetta , a conclusion which may be generalized to the entire study group. © 2002 The Linnean Society of London, Zoological Journal of the Linnean Society , 2002, 134 , 317–334.     

Unravelling evolutionary lineages among South African velvet worms (Onychophora: Peripatopsis) provides evidence for widespread cryptic speciation

The endemic South African velvet worm genus Peripatopsis currently contains eight recognized species described from variable morphological characters and the current taxonomy is unsatisfactory. In an attempt to investigate evolutionary relationships within Peripatopsis, we collected 137 individuals from 34 sample localities for six of the eight species. Sequence data derived from two partial mitochondrial (mt)DNA gene loci (COI and 12S rRNA), as well as partial sequence data from the ribosomal nuclear 18S rDNA locus in combination with gross morphological characters and scanning electron microscopy (SEM), was used to examine evolutionary relationships. Phylogenetic relationships were investigated using minimum evolution (ME) and Bayesian inferences (BI). Additionally, we also undertook a maximum likelihood (ML) analyses on the combined DNA sequence data set. The combined DNA evidence topologies derived from the ME, BI, and ML was highly congruent and was characterized by the presence of multiple lineages within recognized taxa. Peripatopsis clavigera, Peripatopsis moseleyi, and Peripatopsis sedgwicki each comprised two evolutionary lineages; Peripatopsis capensis comprised three; and Peripatopsis balfouri comprised six operational taxonomic units respectively. Genealogical exclusivity at both mtDNA and nuclear DNA among the geographically coherent groups coupled with pronounced sequence divergence suggested a two‐fold increase in the number of species within Peripatopsis. Previously used gross morphological characters (such as the number of leg pairs and colour) were either highly variable within operational taxonomic units, or were invariant, suggesting that alternative morphological characters are necessary for species discrimination. SEM results revealed potentially useful diagnostic characters that can discriminate between at least discriminate some of the newly‐identified lineages. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society 2009, 97 , 200–216.     

Phylogeny and evolution of the South African genus Metalasia (Asteraceae–Gnaphalieae) inferred from molecular and morphological data

Metalasia is a genus in tribe Gnaphalieae (Asteraceae), endemic to South Africa and with its main distribution in the Cape Floristic Region. The genus comprises 57 species and, with a number of closely related genera, it constitutes the ‘Metalasia clade’. A species‐level phylogenetic analysis is presented, based on DNA sequences from two nuclear (internal and external transcribed spacer: ITS, ETS) and two plastid (psbA‐trnH, trnL‐trnF) regions together with morphological data. Analyses combining molecular and morphological data attempt not only to resolve species interrelationships, but also to detect patterns in character evolution. Phylogenetic analyses corroborate our earlier study and demonstrate that Metalasia is formed of two equally sized, well‐supported sister groups, one of which is characterized by papillose cypselas. The results differ greatly from earlier hypotheses based on morphology alone, as few morphological characters support the phylogenetic patterns obtained. The two clades of Metalasia do, however, appear to differ in distribution, corresponding to the different rainfall regimes of South Africa. Analyses show a few taxa to be problematic; one example is the widely distributed M. densa which appears to be an intricate species complex. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 173–198.     

Phylogeny of Glaucosomatidae inferred from molecular evidence

Most species of glaucosomatids (Teleostei: Glaucosomatidae) are endemic to Australia, except Glaucosoma buergeri that is widely distributed from Australia to Japan. This study elucidated phylogenetic relationships among glaucosomatids based on the morphological characters of the saccular‐otolith sagitta, in addition to molecular evidence of mitochondrial 16S rDNA, cytochrome oxidase I (COI) and cytochrome b (cyt b) sequences, and nuclear rhodopsin sequences. The topologies of individuals' phylogenetic trees, based on 16S rDNA, COI and cyt b sequences, were statistically indistinguishable from one another, and were only slightly different from a tree based on rhodopsin sequences. These molecular tree topologies, however, differed from species relationships in morphology‐based phylogenetic hypothesis proposed in previous studies. Specimens of G. buergeri from Australia and Taiwan showed differences in the sagitta and molecular differentiation at the four genes, suggesting a possible speciation event. Both molecular and morphological evidences indicate that Glaucosoma magnificum is the plesiomorphic sister species of other glaucosomatid species. Glaucosoma hebraicum is the sister species of a clade composed of G. buergeri and Glaucosoma scapulare. Molecular and morphological evidences also support the species status of G. hebraicum.     

With eyes wide open: a revision of species within and closely related to the Pocillopora damicornis species complex (Scleractinia; Pocilloporidae) using morphology and genetics

Molecular studies have been instrumental for refining species boundaries in the coral genus Pocillopora and revealing hidden species diversity within the extensively studied global species Pocillopora damicornis. Here we formally revise the taxonomic status of species closely related to and within the P. damicornis species complex, taking into account both genetic evidence and new data on morphometrics, including fine‐scale corallite and coenosteum structure. We found that mitochondrial molecular phylogenies are congruent with groups based on gross‐morphology, therefore reflecting species‐level differentiation. However, high levels of gross morphological plasticity and shared morphological characteristics mask clear separation for some groups. Fine‐scale morphological variation, particularly the shape and type of columella, was useful for differentiating between clades and provides an excellent signature of the evolutionary relationships among genetic lineages. As introgressive hybridization and incomplete lineage sorting complicate the delineation of species within the genus on the basis of a single species concept, the Unified Species Concept may represent a suitable approach in revising Pocillopora taxonomy. Eight species are herein described (P. damicornis, P. acuta, P. aliciae, P. verrucosa, P. meandrina, P. eydouxi, P. cf. brevicornis), including a novel taxon – P ocillopora bairdi sp. nov. (Schmidt‐Roach, this study). Citation synonyms and type materials are presented. © 2014 The Linnean Society of London     

Densely sampled phylogenetic analyses of the Lesser Short-toed Lark (Alaudala rufescens) — Sand Lark (A. raytal) species complex (Aves,Passeriformes) reveal cryptic diversity

The taxonomy of the Lesser/Asian Short-toed Lark Alaudala rufescens–cheleensis complex has been debated for decades, mainly because of minor morphological differentiation among the taxa within the complex, and different interpretations of the geographical pattern of morphological characters among different authors. In addition, there have been few studies based on non-morphological traits. It has recently been suggested based on a molecular study of the lark family Alaudidae that the Sand Lark A. raytal is nested within this complex. We here analysed mitochondrial cytochrome b (cyt b) from 130 individuals across the range of this complex (hereafter called Alaudala rufescens–raytal complex), representing all except two of the 18 currently recognized subspecies. We also analysed 11 nuclear markers from a subsample of these individuals, representing all of the clades found in the cyt b tree. Five primary clades were recovered, which confirmed that A. raytal is nested within this complex. Divergence time estimates among these five clades ranged from 1.76 to 3.16 million years (my; 95% highest posterior density [HPD] 1.0–4.51 my) or 1.99–2.53 my (95% HPD 0.96–4.3 my) in different analyses. Only four of the currently recognized subspecies were recovered as monophyletic in the cyt b tree. Our results call for a taxonomic revision, and we tentatively suggest that at least four species should be recognized, although we stress the need for an approach integrating molecular, morphological and other data that are not yet available.     

The impact of the Congo River and its tributaries on the rodent genus Praomys: speciation origin or range expansion limit?

We studied the taxonomy, distribution, and ecology of species within Praomys, a common rodent genus present in rainforests and montane forests in sub‐Saharan Africa. The taxonomy of the group is problematic, and for the sampled region of Kisangani (Democratic Republic of Congo) no prior genetic study has been published. We used a combination of molecular (cyt b sequencing) and craniometric techniques (canonical analyses of skull measurements) for the species identification of a total of 654 specimens. We confirm the presence of Praomys minor in the region, up to now only known from the type and paratype specimens. At least seven species are present in the Kisangani region, and two clades occur along both banks of the Congo River. The present‐day distribution of the genus seems to be influenced by large‐scale rainforest fragmentation related to drier periods in geological history. The Congo River could in this case constitute a modern barrier to gene flow when the climate enabled rainforest expansion. The tributaries of the Congo River play no role in limiting Praomys species distribution, apart from the Aruwimi River for Praomys jacksoni s.l. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 983–1002.     

Phylogenetic signal in the evolution of body colour and spicule skeleton in calcareous sponges

Some of the morphological characters used in Porifera taxonomy have often been shown to be inconsistent. In the present study, we tested the phylogenetic coherence of currently used taxonomic characters of the calcarean genus Clathrina. For this, 20 species of Clathrina and three other calcinean genera (Ascandra, Guancha, and Leucetta) were sequenced for the ITS and D2 region of the 28S ribosomal DNA. Maximum‐likelihood and maximum‐parsimony algorithms were used to reconstruct phylogenetic trees. Deep divergences were observed in our tree and Clathrina was shown to be paraphyletic. The major split in our topology showed a clear‐cut distinction between sponges with and without tetractine spicules. Moreover, a group of yellow‐coloured Clathrina was clearly separated from the remaining white‐coloured species. Our results show that the presence of diactines, water‐collecting tubes, the degree of cormus anastomosis, and actine shapes do not correlate with the major clades of the calcinean phylogeny. On the other hand, the presence of tripods, the absence of tetractines, and the presence of spines in the apical actine of tetractines seem to be good synapomorphies for clades in our tree. Our results demonstrate that skeleton characters can be reliably used in higher level taxonomy in Clathrinida. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 1026–1034.     

Resurrection in Carapa (Meliaceae): a reassessment of morphological variation and species boundaries using multivariate methods in a phylogenetic context

The taxonomy of the amphi‐Atlantic tree genus Carapa (Meliaceae) has long been controversial. Of the three species currently recognized in the genus, two are known to present substantial morphological variation that has been used in the past to distinguish several taxa, most of which are currently placed in synonymy. Here, a combination of field observations, univariate analyses of leaf, floral and seed characters and principal coordinate analyses of floral characters in the context of a molecular phylogenetic analysis was used to investigate the patterns of variation and delimit morphological species anew in the genus. These results support the recognition of 27 species in Carapa, of which 16 are previously described and 11 are new. In general, phylogenetically related species occurred in the same geographical area, but were morphologically distinct. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 186–221.     

Phylogeny and taxonomy of the Prenolepis genus‐group of ants (Hymenoptera: Formicidae)

Abstract. We investigated the phylogeny and taxonomy of the Prenolepis genus‐group, a clade of ants we define within the subfamily Formicinae comprising the genera Euprenolepis, Nylanderia, gen. rev. , Paraparatrechina, gen. rev. & stat. nov. , Paratrechina, Prenolepis and Pseudolasius. We inferred a phylogeny of the Prenolepis genus‐group using DNA sequence data from five genes (CAD, EF1αF1, EF1αF2, wingless and COI) sampled from 50 taxa. Based on the results of this phylogeny the taxonomy of the Prenolepis genus‐group was re‐examined. Paratrechina (broad sense) species segregated into three distinct, robust clades. Paratrechina longicornis represents a distinct lineage, a result consistent with morphological evidence; because this is the type species for the genus, Paratrechina is redefined as a monotypic genus. Two formerly synonymized subgenera, Nylanderia and Paraparatrechina, are raised to generic status in order to provide names for the other two clades. The majority of taxa formerly placed in Paratrechina, 133 species and subspecies, are transferred to Nylanderia, and 28 species and subspecies are transferred to Paraparatrechina. In addition, two species are transferred from Pseudolasius to Paraparatrechina and one species of Pseudolasius is transferred to Nylanderia. A morphological diagnosis for the worker caste of all six genera is provided, with a discussion of the morphological characters used to define each genus. Two genera, Prenolepis and Pseudolasius, were not recovered as monophyletic by the molecular data, and the implications of this result are discussed. A worker‐based key to the genera of the Prenolepis genus‐group is provided.     

Taxonomic significance of leaf surface morphology in Aloe section Pictae (Xanthorrhoeaceae)

Leaf surface morphology was analysed in 32 species representing the maculate species complex (the poorly resolved section Pictae) in the genus Aloe (Xanthorrhoeaceae). Few comparative morphological data are available for the complex. Leaf surface and stomatal characters observed by scanning electron microscopy show taxonomically significant interspecific variation. Most species are characterized by irregularly outlined, four‐ to six‐sided epidermal cells, the periclinal walls of which are flat and embellished with micropapillae and the anticlinal walls of which are indicated by channels on the leaf surface. The outer stomatal pore is typically sunken or plane and surrounded by four lobes on the leaf surface that may overarch the epistomatal chamber. The guard cells have distinct outer and inner stomatal ledges. Two geographical groups, comprising southern and east African species, are distinguishable by their leaf surface morphology. These characters are diagnostic in A. ellenbeckii, A. prinslooi and A. suffulta and support changes in the delimitation of A. greatheadii, A. macrocarpa and A. swynnertonii. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 418–428.     

Assessing the Biodiversity of Monoraphidium Using 18S rDNA Sequences

The taxonomy of the genus Monoraphidium is unclear due in part to the absence of morphological features to clearly distinguish one species from another. Phytoplankton samples collected from lakes in the Arrowwood National Refuge in eastern North Dakota were found to contain several morphological species of Monoraphidium. Eighteen Monoraphidium isolates were examined with light microscopy and six morphological species were identified. PCR–RFLP of the 18S rDNA was used to type the isolates. Following digestion by Hae III and Taq I, the 18S rDNA PCR–RFLP patterns indicated 10 different types. Presently, the 18S rDNA product is being sequenced for each of the 10 types. By examining morphological characters and 18S rDNA sequences, congruence between morphology and sequence data may be compared. Also, because there is a lack of morphological characters defining Monoraphidium species, diversity within the 18S rDNA sequences may aid in the taxonomy of the genus and its place within the Chlorococcales. Supported by National Science Foundation Grants MCB‐0084188 and DBI‐0070387.     

Taxonomy and molecular phylogeny of the Neotropical genus Atlantoscia (Oniscidea,Philosciidae): DNA barcoding and description of two new species

Identification to the species level using morphology is challenging when usual diagnostic characters are similar amongst related taxa. Within the Neotropical genus Atlantoscia, differences between nominal species are generally small and restricted to a few characters. Despite the power of DNA sequencing to identify and distinguish between species, molecular phylogenies of terrestrial isopods from the Neotropics have not been determined. In this study, two new species of Atlantoscia were described and molecular markers were used to verify both the validity of the current taxonomy and the relationships amongst the species within this genus. All of the recognized Atlantoscia species were strongly supported in the generated phylogenetic trees. The average congeneric distance was 14.7%, with Atlantoscia ituberasensis and Atlantoscia rubromarginata showing the highest genetic divergence. Our results demonstrate the utility of the cytochrome c oxidase subunit I gene together with DNA barcoding to reliably distinguish Atlantoscia species. They also show that DNA barcoding may be helpful in those cases in which classical taxonomy does not provide clear‐cut species resolution. © 2015 The Linnean Society of London     

New insights into the phylogenetics and biogeography of Arum (Araceae): unravelling its evolutionary history

The heat‐ and odour‐producing genus Arum (Araceae) has interested scientists for centuries. This long‐term interest has allowed a deep knowledge of some complex processes, such as the physiology and dynamics of its characteristic lure‐and‐trap pollination system, to be built up. However, mainly because of its large distributional range and high degree of morphological variation, species' limits and relationships are still under discussion. Today, the genus comprises 28 species subdivided into two subgenera, two sections and six subsections. In this study, the phylogeny of the genus is inferred on the basis of four plastid regions, and the evolution of several morphological characters is investigated. Our phylogenetic hypothesis is not in agreement with the current infrageneric classification of the genus and challenges the monophyly of several species. This demonstrates the need for a new infrageneric classification based on characters reflecting the evolution of this enigmatic genus. To investigate the biogeography of Arum deeply, further spatiotemporal analyses were performed, addressing the importance of the Mediterranean basin in the diversification of Arum. Our results suggest that its centre of origin was the European–Aegean region, and that major diversification happened during the last 10 Myr. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163 , 14–32.     

Integrative insect taxonomy based on morphology,mitochondrial DNA,and hyperspectral reflectance profiling

Integrative taxonomy is considered a reliable taxonomic approach of closely related and cryptic species by integrating different sources of taxonomic data (genetic, ecological, and morphological characters). In order to infer the boundaries of seven species of the evacanthine leafhopper genus Bundera Distant, 1908 (Hemiptera: Cicadellidae), an integrated analysis based on morphology, mitochondrial DNA, and hyperspectral reflectance profiling (37 spectral bands from 411–870 nm) was conducted. Despite their morphological similarities, the genetic distances of the cytochrome c oxidase subunit I (COI) gene among the tested species are relatively large (5.8–17.3%). The species‐specific divergence of five morphologically similar species (Bundera pellucida and Bundera spp. 1–4) was revealed in mitochondrial DNA data and reflectance profiling. A key to identifying males is provided, and their morphological characters are described. Average reflectance profiles from the dorsal side of specimens were classified based on linear discriminant analysis. Cross‐validation of reflectance‐based classification revealed that the seven species could be distinguished with 91.3% classification accuracy. This study verified the feasibility of using hyperspectral imaging data in insect classification, and our work provides a good example of using integrative taxonomy in studies of closely related and cryptic species. © 2015 The Linnean Society of London     

Molecular Phylogeny of the Chipmunks Inferred from Mitochondrial Cytochrome b and Cytochrome Oxidase II Gene Sequences

There are currently 25 recognized species of the chipmunk genus Tamias. In this study we sequenced the complete mitochondrial cytochrome b (cyt b) gene of 23 Tamias species. We analyzed the cyt b sequence and then analyzed a combined data set of cyt b along with a previous data set of cytochrome oxidase subunit II (COII) sequence. Maximum-likelihood was used to further test the fit of models of evolution to the cyt b data. Other sciurid cyt b sequence was added to examine the evolution of Tamias in the context of other sciurids. Relationships among Tamias species are discussed, particularly the possibility of a current sorting event among taxa of the southwestern United States and the extreme divergences among the three subgenera (Neotamias, Eutamias, and Tamias).