Determine Species Boundaries Research Articles

Using molecular and morphometric data as operational criteria for the analysis of a threatened rainforest species complex shows interspecific variation, with implications for cryptic-species delimitation and conservation

Resolution of systematic associations and species boundaries is vital for developing conservation priorities for threatened taxa. A complex of Fontainea (Euphorbiaceae) populations, endemic to rainforest communities in central-eastern Australia, comprises several taxonomically challenging species. Fontainea oraria is Critically Endangered, with only one natural population of 10 mature individuals, with the closely related species F. australis being listed as Vulnerable. A recently discovered Fontainea population (currently nominated as F. sp. Coffs Harbour) with taxonomic similarities to F. oraria has been provisionally listed as Critically Endangered, with fewer than 40 individuals. This study employed an integrative approach, combining genetic data and morphometrics to determine species boundaries for three threatened Fontainea population groups as a model system for an integrative approach to delimiting cryptic species. Although our results suggest the potential subdivision of the population groups into three taxa, caution is warranted because this hypothesis remains inconclusive. Taxonomic challenges demand a careful approach, acknowledging the possibility of alternative interpretations. Contrary to supporting distinct species, our morphological and genetic data may also be interpreted as indicative of a single species with geographic variation, a phenomenon observed in genetically linked populations experiencing isolation by distance. Overall, we highlight the need for further research to establish species limits to guide conservation actions.

Determination of the species boundaries of genus Dolerus (Hymenoptera: Tenthredinidae) using the COI gene

New generation molecular approaches and methods are being developed to identify species and determine species boundaries. There are many different approaches of species delimitation used to assess the species richness of poorly studied and highly diverse invertebrate taxa. The basis of these approach is DNA barcoding studies. DNA barcoding has been used as a powerful tool for species identification and delimitation. Although DNA barcoding studies have been carried out on the family Tenthredinidae, there are no studies on species delimitation. Herein, we compare species delimitation analyzes belong to Dolerus genus based on cytochrome c oxidase I (COI) region. In this context, it was used five species delimitation approaches (ABGD, ASAP, DNA Taxon, PTP and GMYC). Thirty-six morphotypes were used in the study. These morphotypes separated into six species (Dolerus triplicatus, Dolerus germanicus, Dolerus puncticollis, Dolerus nigratus, Dolerus sp1 and Dolerus sp2) in ABGD, ASAP and DNA Taxon approaches. Two additional species were introduced because of the tree-based PTP and GMYC approaches. These species were named as Dolerus sp3 and Dolerus sp4 which were separated from Dolerus puncticollis clade and Dolerus nigratus clade, respectively. These analyzes were supported by the phylogenetic tree and CBC entities that constitute the ITS2 data.

Using integrative taxonomy to distinguish cryptic halfbeak species and interpret distribution patterns, fisheries landings, and speciation

Context Species classification disputes can be resolved using integrative taxonomy, which involves the use of both phenotypic and genetic information to determine species boundaries. Aims Our aim was to clarify species boundaries of two commercially important cryptic species of halfbeak (Hemiramphidae), whose distributions overlap in south-eastern Australia, and assist fisheries management. Methods We applied an integrative taxonomic approach to clarify species boundaries and assist fisheries management. Key results Mitochondrial DNA and morphological data exhibited significant differences between the two species. The low level of mitochondrial DNA divergence, coupled with the lack of difference in the nuclear DNA, suggests that these species diverged relatively recently (c. 500 000 years ago) when compared with other species within the Hyporhamphus genus (>2.4 million years ago). Genetic differences between the species were accompanied by differences in modal gill raker counts, mean upper-jaw and preorbital length, and otolith shape. Conclusions On the basis of these genetic and morphological differences, as well as the lack of morphological intergradation between species along the overlapping boundaries of their geographical distributions, we propose that Hyporhamphus australis and Hyporhamphus melanochir remain valid species. Implications This study has illustrated the need for an integrative taxonomic approach when assessing species boundaries and has provided a methodological framework for studying other cryptic fish species in a management context.

Genetic structure of Alhagi (Hedysareae, Fabaceae) populations using ISSR data in Iran.

Alhagi Gagnebin (Fabaceae: Hedysareae) is a small genus of shrubs or subshrubs distributed in temperate and tropical regions of Asia, Europe, and Africa. The exact number of Alhagi species in Iran has been disputable. Studies showed that morphology, seed protein, and chromosome characters could not clearly delimitate the species boundaries of the genus. In order to determine species boundaries and the population structure of Alhagi species in Iran, eight labeled inter simple sequence repeat (ISSR) primers were used to screen 22 populations including 110 individuals. STRUCTURE, PCoA and clustering analyses of ISSR data were able to clearly split all populations of Alhagi in Iran into two distinct groups. As the populations A. pseudalhagi and A. maurorum jointly formed a group (A. maurorum complex) segregated from those of Alhagi graecorum. Further analyses of A. maurorum complex showed a significant molecular difference among the studied populations (PhiPT value = 0.213, P = 0.001) as well as a high amount of variability within populations (79%) indicating frequent genetic exchanges. Structure analysis of complex populations (K = 2) showed no distinct genetic pattern related to their geographical distribution. In this study, Alhagi has retrieved the only two species of Alhagi in Iran; A. graecorum and A. maurorum.

A complement to DNA barcoding reference library for identification of fish from the Northeast Pacific.

The seas of the North Pacific Ocean are characterized by a large variety of fish fauna, including endemic species. Molecular genetic methods, often based on DNA barcoding approaches, have been recently used to determine species boundaries and identify cryptic diversity within these species. This study complements the DNA barcode library of fish from the Northeast Pacific area. A library based on 154 sequences of the mitochondrial COI gene from 44 species was assembled and analyzed. It was found that 39 species (89%) can be unambiguously identified by the clear thresholds forming a barcoding gap. Deviations from the standard 2% threshold value resulted in detection of the species Enophrys lucasi in the sample, which is not typical for the eastern part of the Bering Sea. This barcoding gap also made it possible to identify naturally occurring low values of interspecific divergence of eulittoral taxa Aspidophoroides and the deep-sea genus Coryphaenoides. Synonymy of the genus Albatrossia in favor of the genus Coryphaenoides is suggested based on both the original and previously published data.

Species Boundaries and Parapatric Speciation in the Complex of Alpine Shrubs, Rosa sericea (Rosaceae), Based on Population Genetics and Ecological Tolerances.

Discerning species boundaries among closely related taxa is fundamental to studying evolution and biodiversity. However, species boundaries can be difficult to access in plants because ongoing divergence and speciation may leave an evolutionary footprint similar to introgression, which occurs frequently among species and genera. In this study, we sought to determine species boundaries between two closely related alpine shrubs, Rosa sericea and Rosa omeiensis, using population genetics, environmental data and ecological niche modeling, and morphological traits. We analyzed populations of R. sericea and R. omeiensis using genetic markers comprising a fragment of the single-copy nuclear gene, LEAFY, micro-satellites (EST-SSR), and plastid DNA sequences. The DNA sequence data suggested clusters of populations consistent with geography but not with previously proposed species boundaries based on morphology. Nevertheless, we found that the ecological niches of the previously proposed species only partially overlap. Thus, we suspect that these species are in the process of parapatric speciation; that is, differentiating along an ecological gradient, so that they exhibit differing morphology. Morphology has previously been the basis of recognizing the species R. sericea and R. omeiensis, which are the most widely distributed species within a broader R. sericea complex that includes several other narrow endemics. Here, we recognize R. sericea and R. omeiensis as independent species based on morphological and ecological data under the unified species concept, which emphasizes that these data types are of equal value to DNA for determining species boundaries and refining taxonomic treatments. While the DNA data did not delimit species within the R. sericea complex, we expect to develop and utilize new, robust DNA tools for understanding speciation within this group in future studies.

A COI DNA barcoding survey of Pratylenchus species in the Great Plains Region of North America

Abstract Pratylenchus species are among the most common plant parasitic nematodes in the Great Plains Region of North America. Our goal was to survey Pratylenchus species diversity across the Great Plains region using a mitochondrial COI DNA barcode. The objectives were to (i) determine species boundaries of the common Pratylenchus species within the region, (ii) assess the host associations of the barcoded Pratylenchus specimens, and (iii) determine Pratylenchus distribution patterns throughout the region. A total of 860 soil samples, primarily associated with eight major crops, were collected from Colorado, Kansas, Montana, Nebraska, North Dakota, and Wyoming. From this total, 246 soil samples provided the majority of 915 individual nematode specimens that were amplified by PCR and sequenced for a 727 to 739 bp region of COI. Maximum likelihood, neighbor-joining, and Bayesian phylogenetic trees all recognized 19 distinct and well-supported haplotype groups. The most common and widespread haplotype group, representing 53% of all specimens was P. neglectus, detected from 178 fields in 100 counties and associated with fields growing wheat, corn, dry beans, barley, alfalfa, sugar beets, potatoes, and a vineyard. The second most prevalent haplotype group was P. scribneri, representing 30% of all specimens and recovered from 104 fields in 45 counties, and most frequently associated with corn. Mixed field populations were encountered in approximately 20% of infested fields, with P. neglectus and P. scribneri most often occurring together in corn-soybean cropping systems. Less frequently encountered from the region were P. hexincisus, P. thornei, P. penetrans, P. alleni, and P. zeae. Eight additional haplotype groups, potentially new Pratylenchus species, were discovered in the survey.

Molecular identification of three species of Oncideres (Coleoptera: Cerambycidae) using RAPD markers.

Twig girdlers have a short adult life and a long larval stage (up to one year in some species). This fact, together with lack of morphological traits to identify the species from the larval stage, poses obstacles for the taxonomic identification and characterization of the many twig girdlers found in the wild. To solve this matter, Random Amplified Polymorphic DNA (RAPD) molecular markers have been applied to identify some insect species and to determine species boundaries. The aim of this study was to identify three species of Oncideres using the RAPD technique. Adults and larvae of O. saga, O. ocularis and O. ulcerosa were collected in Minas Gerais, Brazil. Two RAPD primers were used (OPA-05 and OPB-13), which produced different electrophoretic profiles that were used to construct a UPGMA phenogram. Three groups identified in the analyses accurately separated the three species, based on individuals in two different stages of development (adults and larvae): I: O. saga, II: O. ulcerosa, and III: O. ocularis. Molecular markers, such as RAPD, are valuable tools that help taxonomists in species identification.

New insight into the species diversity and life cycles of rust fungi (Pucciniales) affecting bioenergy switchgrass (Panicum virgatum) in the Eastern and Central United States

Research was undertaken to clarify the taxonomic identity of leaf rust (Pucciniales) fungi on bioenergy switchgrass in the Eastern and Central U.S. We integrated internal transcribed spacer 2 (ITS2) and partial 28S ribosomal RNA gene sequence data from collections taken from cultivated switchgrass and herbarium specimens, including purported aecial and telial states of Puccinia graminicola and Puccinia pammelii. Maximum likelihood and Bayesian analyses revealed four monophyletic clades: Puccinia emaculata sensu stricto (s.s.), P. pammelii, P. graminicola, and Puccinia novopanici. Results also indicated that P. emaculata s.s. was not affecting cultivated, bioenergy switchgrass. Aecidium pammelii and P. pammelii were distinct phylogenetically from P. emaculata s.s. and grouped within a well-supported clade, demonstrating aecial-telial host alternation for P. pammelii between Euphorbia corollata and switchgrass. Aecidium stillingiae on queen’s delight (Stillingia sylvatica)—a purported aecial state host for P. graminicola—shared identical sequences with the recently described species Puccinia pascua. The latter fungus, however, was recovered within a subclade of P. graminicola. Hence, queen’s delight likely is not an aecial host to P. graminicola s.s. Additional molecular studies are warranted to determine species boundaries within the P. graminicola complex. The majority of contemporary collections from cultivated switchgrass were recognized as P. novopanici. Collectively, bioenergy switchgrass is host to at least three phylogenetically distinct species, presenting a significant challenge to the future selection and breeding of switchgrass with improved rust resistance.

Lack of host specificity of copepod crustaceans associated with mushroom corals in the Red Sea

The radiation of symbiotic copepods (Crustacea: Copepoda) living in association with stony corals (Cnidaria: Scleractinia) is considered host-specific and linked to the phylogenetic diversification of their hosts. However, symbiotic copepods are poorly investigated, occurrence records are mostly anecdotal, and no explicit analysis exists regarding their relationship with the hosts. Here, we analysed the occurrence of symbiotic copepods on different co-occurring and phylogenetically closely related scleractinian corals. We used an innovative approach of DNA extraction from single microscopic specimens that preserves the shape of the organisms for integrative morphological studies. The rationale of the study involved: (i) sampling of mushroom corals (Fungiidae) belonging to 13 species and eight genera on different reefs along the Saudi coastline in the Red Sea, (ii) extraction of all the associated copepods, (iii) morphological screening and identification of copepod species, (iv) use of DNA taxonomy on mitochondrial and nuclear markers to determine species boundaries for morphologically unknown copepod species, (v) reconstruction of phylogenies to understand their evolutionary relationships, and (vi) analysis of the ecological drivers of the occurrence, diversity and host specificity of the copepods. The seven species of coral-associated copepods, all new to science, did not show any statistically significant evidence of host-specificity or other pattern of ecological association. We thus suggest that, contrary to most assumptions and previous anecdotal evidence on this coral-copepod host-symbiont system, the association between copepods and their host corals is rather labile, not strict, and not phylogenetically constrained, changing our perception on evolutionary patterns and processes in symbiotic copepods.

How many names for a beloved genus? – Coalescent-based species delimitation in Xanthium L. (Ambrosiinae, Asteraceae)

The species category is the fundamental unit in biology. In the last decades, several studies have been carried out, using sequence information and phylogenies to resolve issues in taxonomically problematic groups. The multispecies coalescent theory, and the species-delimitation methods developed in the last years based on that, offer powerful and objective tools to determine species boundaries using sequence data.The genus Xanthium is a morphologically variable complex with several local forms, nowadays become cosmopolitan due to human-mediated dispersal. Past taxonomic treatments of the genus were based essentially on the burr morphology. They varied considerably in number of recognized species, depending on the importance that the different authors gave to burr traits such as burr size, pubescence, number and length of spines in the burrs, and the degree to which those are hooked.We used sequence information from two plastid regions (the intergenic spacer regions psbA-trnH and trnQ-rps16) and two nuclear ones (ITS1-5.8 S-ITS2 rDNA, and the single-copy nuclear marker D35). Two of the most advanced coalescent-based species delimitation methods (BP&P and STACEY) were applied, in order to objectively determine species boundaries in Xanthium.Results from the species-delimitation methods strongly support scenarios with a reduced number of species. Prior on the effective population size parameter (θ) had a strong influence on BP&P results. Analyses with large and small θ prior supported species delimitation scenarios with four and five species, respectively. STACEY recognized five cluster in the dataset, all supported by high posterior probability values. Five species are identified in Xanthium, in a great extent corresponding to the infrageneric classification given for the genus in past taxonomic revisions.

Delimiting species of Protaphorura (Collembola: Onychiuridae): integrative evidence based on morphology, DNA sequences and geography

Species delimitation remains a significant challenge when the diagnostic morphological characters are limited. Integrative taxonomy was applied to the genus Protaphorura (Collembola: Onychiuridae), which is one of most difficult soil animals to distinguish taxonomically. Three delimitation approaches (morphology, molecular markers and geography) were applied providing rigorous species validation criteria with an acceptably low error rate. Multiple molecular approaches, including distance- and evolutionary model-based methods, were used to determine species boundaries based on 144 standard barcode sequences. Twenty-two molecular putative species were consistently recovered across molecular and geographical analyses. Geographic criteria were was proved to be an efficient delimitation method for onychiurids. Further morphological examination, based on the combination of the number of pseudocelli, parapseudocelli and ventral mesothoracic chaetae, confirmed 18 taxa of 22 molecular units, with six of them described as new species. These characters were found to be of high taxonomical value. This study highlights the potential benefits of integrative taxonomy, particularly simultaneous use of molecular/geographical tools, as a powerful way of ascertaining the true diversity of the Onychiuridae. Our study also highlights that discovering new morphological characters remains central to achieving a full understanding of collembolan taxonomy.

High morpho‐anatomical variability in Halimeda macroloba (Bryopsidales, Chlorophyta) in Thai waters

SUMMARYHalimeda macroloba is a common widespread and morphologically variable species in the Indo‐Pacific Ocean. A series of morphometric data (16 morphological and 46 anatomical variables) and the chloroplast‐encoded tuf A gene were examined to explore the morphological and genetic variations within the species and to better determine species boundaries in H. macroloba in Thai waters. Moreover, the environmental conditions, which may affect morphological variations in H. macroloba, especially of Haplotype I were examined. Our results showed that H. macroloba has more morpho‐anatomical variability and broader species boundaries than previously thought in several characters: holdfast type, segment shape and size, node height with differences in pore size, the presence of two additional patterns of peripheral utricle as seen in surface view, and additional layers of utricles (up to seven layers). Additionally, two new variable characteristics have been reported for the first time, i.e. shape and margin of peripheral utricles. Water depth and light correlated with thallus morphology, larger thalli and segments with a greater number of utricle layers were associated with subtidal plants, while smaller thalli and segments were found in the intertidal or high light intensity areas. The high plasticity of this species may explain why in the Indo‐Pacific Ocean this species is common and dominant.

Revision of the Nearctic species of Callomyia Meigen (Diptera: Platypezidae) and phylogeny of the genus.

The Nearctic fauna of the genus Callomyia Meigen is revised and a phylogeny of the world species, based on morphological characters, is presented. Although morphological data are used primarily to delimit species, molecular sequence data (DNA barcodes) are used where possible, to help determine species boundaries and associate sexes. Species descriptions, diagnoses, and distribution maps are presented, along with illustrations of habitus, male terminalia, and additional important diagnostic characters. A key to the Nearctic species is provided. Ten species are recorded from the Nearctic Region including three new species: C. argentea Cumming sp. nov., C. arnaudi Cumming sp. nov., C. bertae Kessel, C. browni Cumming sp. nov., C. calla Kessel, C. corvina Kessel, C. gilloglyorum Kessel, C. proxima Johnson, C. velutina Johnson, and C. venusta Snow. The female of C. velutina is described, and three new synonyms are proposed: C. cleta Kessel is a junior synonym of C. calla syn. nov.; C. clara Kessel is a junior synonym of C. corvina syn. nov.; and C. liardia Kessel & Buegler is a junior synonym of C. proxima syn. nov. Phylogenetic relationships within the genus are reconstructed. The genus is monophyletic based primarily on the setulose R₁wing vein, female antennal size and three larval characters. The Nearctic species do not form a monophyletic group with respect to the Old World species.

Cryptic diversity, high host specificity and reproductive synchronization in army ant-associated Vatesus beetles.

Army ants and their arthropod symbionts represent one of the most species-rich animal associations on Earth, and constitute a fascinating example of diverse host-symbiont interaction networks. However, despite decades of research, our knowledge of army ant symbionts remains fragmentary due to taxonomic ambiguity and the inability to study army ants in the laboratory. Here, we present an integrative approach that allows us to reliably determine species boundaries, assess biodiversity, match different developmental stages and sexes, and to study the life cycles of army ant symbionts. This approach is based on a combination of community sampling, DNA barcoding, morphology and physiology. As a test case, we applied this approach to the staphylinid beetle genus Vatesus and its different Eciton army ant host species at La Selva Biological Station, Costa Rica. DNA barcoding led to the discovery of cryptic biodiversity and, in combination with extensive community sampling, revealed strict host partitioning with no overlap in host range. Using DNA barcoding, we were also able to match the larval stages of all focal Vatesus species. In combination with studies of female reproductive physiology, this allowed us to reconstruct almost the complete life cycles of the different beetle species. We show that Vatesus beetles are highly adapted to the symbiosis with army ants, in that their reproduction and larval development are synchronized with the stereotypical reproductive and behavioural cycles of their host colonies. Our approach can now be used to study army ant-symbiont communities more broadly, and to obtain novel insights into co-evolutionary and ecological dynamics in species-rich host-symbiont systems.

Species delimitation in plants using the Qinghai-Tibet Plateau endemic Orinus (Poaceae: Tridentinae) as an example.

Accurate identification of species is essential for the majority of biological studies. However, defining species objectively and consistently remains a challenge, especially for plants distributed in remote regions where there is often a lack of sufficient previous specimens. In this study, multiple approaches and lines of evidence were used to determine species boundaries for plants occurring in the Qinghai-Tibet Plateau, using the genus Orinus (Poaceae) as a model system for an integrative approach to delimiting species. A total of 786 individuals from 102 populations of six previously recognized species were collected for niche, morphological and genetic analyses. Three plastid DNA regions (matK, rbcL and trnH-psbA) and one nuclear DNA region [internal transcribed space (ITS)] were sequenced. Whereas six species had been previously recognized, statistical analyses based on character variation, molecular data and niche differentiation identified only two well-delimited clusters, together with a third possibly originating from relatively recent hybridization between, or historical introgression from, the other two. Based on a principle of integrative species delimitation to reconcile different sources of data, the results provide compelling evidence that the six previously recognized species of the genus Orinus that were examined should be reduced to two, with new circumscriptions, and a third, identified in this study, should be described as a new species. This empirical study highlights the value of applying genetic differentiation, morphometric statistics and ecological niche modelling in an integrative approach to re-circumscribing species boundaries. The results produce relatively objective, operational and unbiased taxonomic classifications of plants occurring in remote regions.

Discordance between morphological and molecular species boundaries among Caribbean species of the reef sponge Callyspongia.

Sponges are among the most species-rich and ecologically important taxa on coral reefs, yet documenting their diversity is difficult due to the simplicity and plasticity of their morphological characters. Genetic attempts to identify species are hampered by the slow rate of mitochondrial sequence evolution characteristic of sponges and some other basal metazoans. Here we determine species boundaries of the Caribbean coral reef sponge genus Callyspongia using a multilocus, model-based approach. Based on sequence data from one mitochondrial (COI), one ribosomal (28S), and two single-copy nuclear protein-coding genes, we found evolutionarily distinct lineages were not concordant with current species designations in Callyspongia. While C. fallax,C. tenerrima, and C. plicifera were reciprocally monophyletic, four taxa with different morphologies (C. armigera,C. longissima,C. eschrichtii, and C. vaginalis) formed a monophyletic group and genetic distances among these taxa overlapped distances within them. A model-based method of species delimitation supported collapsing these four into a single evolutionary lineage. Variation in spicule size among these four taxa was partitioned geographically, not by current species designations, indicating that in Callyspongia, these key taxonomic characters are poor indicators of genetic differentiation. Taken together, our results suggest a complex relationship between morphology and species boundaries in sponges.

A genome-wide AFLP replacement in a hybrid population derived from two closely related Viola species from contrasting habitats

Hybrid zones formed from genetically and ecologically differentiated species provide an opportunity to examine how genetics and ecology interact to determine species boundaries. In this study, we examined a hybrid zone generated by invasion of shade-loving Viola eizanensis into habitat occupied by its close relative, V. chaerophylloides var. sieboldiana, which prefers more open areas. We used 339 AFLP loci to estimate genomic composition and generation of hybrids, and confirmed maternal inheritance through sequencing of the cpDNA atpI–atpH intergenic spacer region. Phenotypic variation was assessed using leaf thickness (leaf mass/area) and leaf dissection (leaf perimeter/area), which were diversified between species. Genetic analyses showed that the AFLP of hybrid individuals in having maternal origin from V. chaerophylloides var. sieboldiana were replaced by V. eizanensis in response to habitat. Significant correlations were observed between hybrid indices and the two leaf traits. These results suggest that genes of V. eizanensis that are adaptive to shaded habitats are fixed in the hybrids, and that the degree of leaf thickness and dissection is important in adaptation of the hybrids to their light environments.

Revision of basal macropodids from the Riversleigh World Heritage Area with descriptions of new material of Ganguroo bilamina Cooke, 1997 and a new species

The relationship of basal macropodids (Marsupialia: Macropodoidea) from the Oligo-Miocene of Australia have been unclear. Here, we describe a new species from the Bitesantennary Site within the Riversleigh's World Heritage Area (WHA), Ganguroo bites n. sp., new cranial and dental material of G. bilamina, and reassess material previously described as Bulungamaya delicata and 'Nowidgee matrix'. We performed a metric analysis of dental measurements on species of Thylogale which we then used, in combination with morphological features, to determine species boundaries in the fossils. We also performed a phylogenetic analysis to clarify the relationships of basal macropodid species within Macropodoidea. Our results support the distinction of G. bilamina, G. bites and B. delicata, but 'Nowidgee matrix' appears to be a synonym of B. delicata. The results of our phylogenetic analysis are inconclusive, but dental and cranial features suggest a close affinity between G. bilamina and macropodids. Finally, we revise the current understanding of basal macropodid diversity in Oligocene and Miocene sites at Riversleigh WHA.

More Problems Despite Bigger Flowers: Systematics of <I>Cuscuta tinctoria</I> Clade (subgenus <I>Grammica</I>, Convolvulaceae) with Description of Six New Species

Abstract Cuscuta tinctoria clade, the second largest infrageneric group of subgenus Grammica, includes 14 species that are centered in Mexico and adjacent regions, but also two species that are found in Australia, presumably as a result of long-distance dispersal. Flowers, pollen, and stigmas are among the largest in Cuscuta, with plants being xenogamous or facultatively xenogamous. In this clade, the convergent evolution of morphological traits, especially those associated with the calyx and gynoecium/ capsule, has obscured the identity of some species and has hindered previous efforts to determine their limits. Basic morphology, scanning electron microscopy and sequence data from the nuclear internal transcribed spacer (ITS) and the plastid trnL-F region were used to reconstruct the phylogeny, gain a better understanding of the evolutionary history, and determine species boundaries. Overall, species were grouped in five subclades, Based on their morphological and molecular similarity, C. tinctoria, C. a...