Statistical Parsimony Research Articles

Empirical Evidence Supporting Frequent Cryptic Speciation in Epiphyllous Liverworts: A Case Study of the Cololejeunea lanciloba Complex

Cryptic species are frequently recovered in plant lineages, and considered an important cause for divergent of morphological disparity and species diversity. The identification of cryptic species has important implications for the assessment of conservation needs of species aggregates. The mechanisms and processes of the origin of cryptic species diversity are still poorly understand based on the lack of studies especially in context of environment factors. Here we explored evidence for cryptic species within the epiphyllous liverworts Cololejeunea lanciloba complex based on two loci, the plastid trnL-F region and the nuclear ribosomal ITS region. Several analytic approaches were employed to delimit species based on DNA sequence variation including phylogenetic reconstruction, statistical parsimony networks analysis and two recently introduced species delimitation criteria: Rosenberg’s reciprocal monophyly and Rodrigo’s randomly distinct. We found evidence for thirteen genetically distinct putative species, each consisting of more than one haplotype, rather than four morphologically-circumscribed species. The results implied that the highly conserved phenotypes are not congruent with the genetic differentiation, contributing to incorrect assessments of the biodiversity of epiphyllous liverworts. We hypothesize that evolution of cryptic species recovered may be caused by selection of traits critical to the survival in epiphyllous habitats combined with limited developmental options designed in the small body.

Still a one species genus? Strong genetic diversification in the world’s largest living odonate, the Neotropical damselfly Megaloprepus caerulatus

Mesoamerican biodiversity is increasingly threatened by anthropogenic destruction of natural land cover. Habitat degradation and climate change are primary threats to specialized forest odonate species that are important model organisms for forest health and defining conservation units. The extreme niche specialization of Megaloprepus caerulatus, the world’s largest extant odonate, makes it well suited as an indicator for changing environmental conditions. Megaloprepus, which is considered to be a monospecific genus, is highly dependent on old growth forests whose water filled tree holes are limiting reproductive resources for this species. Here, we focus on the question how historical and recent fragmentation events, strong niche conservatism and ecological conditions have affected population dynamics, viability and the species status in this evolutionarily old genus. Two mitochondrial sequence markers (ND1 and 16S rRNA) and a set of microsatellites were used to analyze population structure and genetic diversity of M. caerulatus in the northern part of its distributional range. Results suggested an absence of gene flow and no shared haplotypes among the study populations. Statistical parsimony indicated high sub-structuring among populations with sequence diversity similar to levels found at the species level compared to other odonates. In sum, the genetic data suggest that Megaloprepus may actually consist of more than one species. The taxonomic status of the group should be revised in light of the three distinct genetic clusters found in different forest regions. The results may also allow insights into the impact of recent and historical habitat fragmentation on a strong Neotropical forest restricted insect species.

Confirmatory factory analysis of the Neck Disability Index in a general problematic neck population indicates a one-factor model

Background contextThe Neck Disability Index frequently is used to measure outcomes of the neck. The statistical rigor of the Neck Disability Index has been assessed with conflicting outcomes. To date, Confirmatory Factor Analysis of the Neck Disability Index has not been reported for a suitably large population study. Because the Neck Disability Index is not a condition-specific measure of neck function, initial Confirmatory Factor Analysis should consider problematic neck patients as a homogenous group. PurposeWe sought to analyze the factor structure of the Neck Disability Index through Confirmatory Factor Analysis in a symptomatic, homogeneous, neck population, with respect to pooled populations and gender subgroups. Study designThis was a secondary analysis of pooled data. Patient sampleA total of 1,278 symptomatic neck patients (67.5% female, median age 41 years), 803 nonspecific and 475 with whiplash-associated disorder. Outcome measuresThe Neck Disability Index was used to measure outcomes. MethodsWe analyzed pooled baseline data from six independent studies of patients with neck problems who completed Neck Disability Index questionnaires at baseline. The Confirmatory Factor Analysis was considered in three scenarios: the full sample and separate sexes. Models were compared empirically for best fit. ResultsTwo-factor models have good psychometric properties across both the pooled and sex subgroups. However, according to these analyses, the one-factor solution is preferable from both a statistical perspective and parsimony. The two-factor model was close to significant for the male subgroup (p<.07) where questions separated into constructs of mental function (pain, reading headaches and concentration) and physical function (personal care, lifting, work, driving, sleep, and recreation). ConclusionsThe Neck Disability Index demonstrated a one-factor structure when analyzed by Confirmatory Factor Analysis in a pooled, homogenous sample of neck problem patients. However, a two-factor model did approach significance for male subjects where questions separated into constructs of mental and physical function. Further investigations in different conditions, subgroup and sex-specific populations are warranted.

Comparative phylogeographic study of Hosta sieboldiana and Hosta albomarginata (Asparagaceae) in Japan

We analyzed variations in chloroplast DNA (cpDNA) in the widespread herbaceous species Hosta sieboldiana and Hosta albomarginata across large portions of their geographic ranges in the Japanese archipelago. Our objective was to compare the phylogeographic histories and phylogeographic structures of the two congeneric species in the Japanese archipelago. The location of the study is Japanese archipelago. We sequenced 1380 bp of noncoding cpDNA from 45 populations of H. sieboldiana (n = 362) and 55 populations of H. albomarginata (n = 436) to assess genetic variations within and among populations across almost the entire distributions of the species in Japan. Extant patterns of geographic structure were analyzed using statistical parsimony networks and spatial analysis of molecular variance (SAMOVA). We also used Monmonier's algorithm to detect genetic barriers between regions. Relationships between the populations were examined using a neighbor-joining (NJ) method. Four haplotypes were found for H. sieboldiana, whereas eight haplotypes were identified for H. albomarginata. Total genetic haplotype diversity (hT) and within-population haplotype diversity (hS) for H. sieboldiana were 0.352 and 0.040, respectively, while the values for H. albomarginata were 0.529 and 0.085, respectively. The population differentiations (GST) for H. sieboldiana and H. albomarginata were 0.839 and 0.886, respectively. The SAMOVA analysis revealed two clusters in H. sieboldiana and four clusters in H. albomarginata. Differentiations between and among the clusters were supported by the BARRIER analysis and the NJ tree. We detected differences in the population genetic structure between the two species. We found that H. sieboldiana had lower haplotype diversity than H. albomarginata. These results may be partially explained by the difference in ecological habitats and geographic distributions between the species. Hosta albomarginata is more widely distributed than H. sieboldiana in East Asia including Russia, and this large distribution range would enable more chances to intraspecific gene flow.

Genetic data generated from virus–host complexes obtained by membrane co-immobilization are equivalent to data obtained from tangential filtrate virus concentrates and virus cultures

The sieving and immobilization of virus-host complexes using impact filtration (aka membrane co-immobilization or MCI) is a novel approach to the study of plankton viruses. One of the most interesting characteristics of the method is the possibility of generating data on potential viral hosts without the need of culturing hosts cells. MCI has demonstrated to be useful for studying viruses of picoalgae, but studies comparing data generated by MCI to data obtained by other techniques are lacking. In this work, Ostreococcus virus (OV) and Ostreococcus sp. sequences generated from virus-host complexes obtained by MCI were compared to sequences obtained from tangential filtration (TF) concentrates and virus cultures (VC). Statistical parsimony, phylogenetic analyses, pairwise distance comparisons, and analysis of molecular variance showed that the viral and host sequences obtained by the three methods were highly related to each other, indicating that MCI, TF, and VC produce equivalent results. Minor differences were observed among viral sequences obtained from VC and TF concentrates as well as among host sequences generated from VC and MCI. As discussed in the body of the paper, the divergence observed for cultured cells could be due to selective pressures exerted by culture conditions, whereas the correlate observed for the corresponding viral sequences could obey to a hitchhiking effect.

Diversification of Scrophularia (Scrophulariaceae) in the Western Mediterranean and Macaronesia – Phylogenetic relationships, reticulate evolution and biogeographic patterns

The flora of the Mediterranean region and Macaronesia is characterized by high levels of species diversity and endemism. We examined phylogenetic relationships of Scrophularia within one of its secondary centers of diversity located in the Iberian Peninsula and adjacent Macaronesia. In total, 65 ingroup accessions from 45 species, representing an almost complete sampling of the region, were analyzed using sequences from the internal transcribed spacer region (ITS) and the plastid trnQ-rps16 intergenic spacer. Phylogenetic relationships were inferred using Bayesian inference, maximum likelihood and statistical parsimony networking. Incongruence between datasets was assessed with statistical tests and displayed by split networks. Biogeographic inferences incorporating information from both markers (despite low resolution in some parts of the trees) and all incongruent taxa were accomplished with a novel combination of methods, using trees generated with the taxon duplication approach as input for Bayesian binary MCMC (BBM) analysis as implemented in RASP.Nuclear and chloroplast markers support a clade which comprises the majority of Iberian and Macaronesian species and consists of three subclades. Analyses of the substantial incongruence observed among markers indicate reticulate evolution and suggest that Scrophularia species diversity in this region is largely attributable to hybridization; a combination of both polyploidy and dysploidy in the karyotypic evolution of Western Mediterranean Scrophularia taxa is proposed. Our results provide support for an ancient hybridization event between two widespread lineages, which resulted in an allopolyploid ancestor of the Iberian – Macaronesian group with 2n=58 chromosomes. The ancestor then diverged into the three main lineages present in the Iberian Peninsula, Northern Africa and Macaronesia today. Subsequent interspecific hybridizations at different ploidy levels additionally generated new species. Presumably, hybridization and diversification within the genus in the Western Mediterranean have not been restricted to one particular event, but occurred repeatedly. It can be assumed that the topographical complexity found in the Iberian Peninsula has promoted diversification and hybrid speciation processes in Scrophularia, and that isolation in glacial refugia has preserved recent and ancient lineages. For the Macaronesian taxa, biogeographic analyses support several origins, by colonizations from at least four distinct lineages.

Variacion genetica de Procambarus llamasi (Decapoda: Cambaridae) en la peninsula de Yucatan: evidencia de conectividad en un paisaje fragmentado

Within the family Cambaridae, the genus Procambarus is especially important due to the number of species it contains and its wide distribution in the American Continent. Procambarus ( Austrocambarus ) llamasi is endemic to the Yucatan Peninsula, distributed in epicontinental water bodies, including lakes, marshes and flood plains, which may be relatively isolated. Due to the fragmented nature of its habitat, it might be expected to find highly structured populations with unique haplotypes. To describe the genetic variation of the different populations of P. llamasi and their distribution pattern, a phylogeographic analysis was performed using the mitochondrial COI gene. A 643 base pair fragment was sequenced from 53 individuals of ten populations. Different haplotypes were identified and a haplotype network was built using statistical parsimony for nested clade analysis. A total of 14 variable sites were found, 12 of which were informative; all nucleotide changes were transitions and nucleotide diversity was π = 0.0045. Ten haplotypes were found with a diversity of HD = 0.532, with one of them widely distributed. The nested clade analysis shows that current haplotypes are the result of a contiguous range expansion, suggesting that the source of the expansion was at the southern portion of the peninsula. This may occur due to the seasonal floodings in the region, which can create temporary connections between populations.

Multiple introductions of serotype O foot-and-mouth disease viruses into East Asia in 2010–2011

Foot-and-mouth disease virus (FMDV) is a highly contagious and genetically variable virus. Sporadic introductions of this virus into FMD-free countries may cause outbreaks with devastating consequences. In 2010 and 2011, incursions of the FMDV O/SEA/Mya-98 strain, normally restricted to countries in mainland Southeast Asia, caused extensive outbreaks across East Asia. In this study, 12 full genome FMDV sequences for representative samples collected from the People’s Republic of China (PR China) including the Hong Kong Special Administrative Region (SAR), the Republic of Korea, the Democratic People’s Republic of Korea, Japan, Mongolia and The Russian Federation were generated and compared with additional contemporary sequences from viruses within this lineage. These complete genomes were 8119 to 8193 nucleotides in length and differed at 1181 sites, sharing a nucleotide identity ≥ 91.0% and an amino acid identity ≥ 96.6%. An unexpected deletion of 70 nucleotides within the 5′-untranslated region which resulted in a shorter predicted RNA stem-loop for the S-fragment was revealed in two sequences from PR China and Hong Kong SAR and five additional related samples from the region. Statistical parsimony and Bayesian phylogenetic analysis provide evidence that these outbreaks in East Asia were generated by two independent introductions of the O/SEA/Mya-98 lineage sometime between August 2008 and March 2010. The rapid emergence of these viruses from Southeast Asia highlights the importance of adopting approaches to closely monitor the spread of this lineage that now poses a threat to livestock industries in other regions.

Did the parasitoid Pnigalio mediterraneus (Hymenoptera: Eulophidae) track the invasion of the horse chestnut leafminer?

How communities of natural enemies, such as parasitoids, adapt to the range expansion of their hosts or the arrival of a novel invasive host is an important question in invasion biology. Do parasitoids track the expansion of their hosts from their shared native range? Do they locally adapt both behaviorally and physiologically to the arrival of a novel species by shifting hosts? Few studies have addressed those questions, yet they are important to develop efficient methods to manage invasive species. Here we focus on Pnigalio mediterraneus Ferriere and Delucchi (Hymenoptera: Eulophidae), an important parasitoid of two major agricultural and ornamental pests, the olive fruit fly Bactrocera oleae Rossi (Diptera: Tephritidae) and the horse chestnut leafminer Cameraria ohridella Deschka & Dimic (Lepidoptera: Gracillariidae). C. ohridella recently invaded Europe starting from the Southern Balkans, whereas B. oleae has been associated since the Quaternary with wild olives in the Mediterranean, where it largely spread after the domestication of cultivated olives. We used two markers, the ribosomal spacer ITS2 and the mitochondrial gene COI. Although the ITS2 dataset provided little variation and no phylogeographic signal, analysis of mtDNA of 188 individuals of P. mediterraneus from 54 European localities allowed us to identify 53 haplotypes. Both nucleotide and haplotype diversity were higher for Mediterranean samples, and from samples reared from B. oleae. The statistical parsimony network identified one haplotype as the most frequent, ancestral and mainly associated with C. ohridella. Our findings suggest that P. mediterraneus locally host switched to C. ohridella from other hosts in the Balkans and later tracked the horse chestnut leafminer invasion over Europe. Therefore both host-tracking and ecological sorting could explain the current distribution of P. mediterraneus haplotypes.

Capturing the population structure of microparasites: using ITS‐sequence data and a pooled DNA approach

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is a common marker not only for the molecular identification of different taxa and strains, but also for the analysis of population structure of wild microparasite communities. Importantly, the multicopy nature of this region allows the amplification of low-quantity samples of the target DNA, a common problem in studies on unicellular, unculturable microparasites. We analysed ITS sequences from the protozoan parasite Caullerya mesnili (class Ichthyosporea) infecting waterflea (Daphnia) hosts, across several host population samples. We showed that analysing representative ITS-types [as identified by statistical parsimony network analysis (SPN)] is a suitable method to address relevant polymorphism. The spatial patterns were consistent regardless of whether parasite DNA was extracted from individual hosts or pooled host samples. Remarkably, the efficiency in detecting different sequence types was even higher after sample pooling. As shown by simulations, aneasily manageable number of sequences from pooled DNA samples are sufficient to resolve the spatial population structure in this system. In summary, the ITS region analysed from pooled DNA samples can provide valuable insights into the spatial and temporal dynamics of microparasites. Moreover, the application of SPN analysis is a good alternative to the well-established neighbour-joining method (NJ) for the identification of representative ITS-types. SPN can even outperform NJ by joining most of the singleton sequences to representative sequence clusters.

Cryptic diversity, intraspecific phenetic plasticity and recent geographical translocations inBranchiomma(Sabellidae, Annelida)

The importance of identifying biological diversity accurately and efficiently is becoming more evident. It is therefore critical to determine the species boundaries between closely related taxa and to establish diagnostic characters that allow us to define species. This is not an easy task when species exhibit high intraspecific phenotypic plasticity or when distinct evolutionary lineages with an unusually large amount of genetic distinctiveness show no apparent morphological diversity (cryptic species). These phenomena appear to be common in the genus of fan wormsBranchiomma(Sabellidae, Annelida), and consequently, taxonomic errors are widespread in the group. Moreover, someBranchiommaspecies have been unintentionally translocated outside the area where natural range extension is expected, increasing the taxonomic problems. We have performed a range of analytical methods including genetic distances, Bayesian inference, maximum likelihood, maximum parsimony, statistical parsimony analyses and general mixed Yule coalescent model to clarify the taxonomic status and assess the species boundaries ofBranchiommain Australia. This study shows that the traditional diagnostic morphological features are greatly homoplastic. Results also indicate that the diversity ofBranchiommain Australia is higher than previously reported and evidence some cases of high phenetic plasticity (in features previously considered as stable within species), high intraspecific genetic variability, cryptic species and several unexpected cases of translocations.

Origin and evolution of the Italian subterranean termiteReticulitermes lucifugus(Blattodea, Termitoidae, Rhinotermitidae)

The Holarctic genus Reticulitermes shows seven species within the Mediterranean Basin. While phylogeny and systematics at continental level has been deeply investigated, a few studies concentrated on local ranges. To gain a clearer picture of the diversity and evolution of the Italian species Reticulitermes lucifugus, we analyzed the mitochondrial cytochrome oxidase II (COII) gene marker in newly collected colonies across the Peninsula. Data were gathered with all R. lucifugus sequences available from previous studies; COII sequences of the closely related Iberian taxa were also added to the data set. Maximum-likelihood, median-joining and statistical parsimony network elaborations on the resulting 119 colonies all agreed in indicating that: (i) the Sardo-Corsican subspecies R. lucifugus corsicus, strictly related to Southern Italian populations (including the Sicilian ones), is phylogenetically closer to the Iberian Reticulitermes grassei; and (ii) R. lucifugus lucifugus peninsular populations are structured into three clusters. The phylogenetic relationships and the biogeography of extant taxa suggest a scenario in which R. lucifugus ancestors colonized the Italian region through the Sardo-Corsican microplate during its Oligocene-Miocene anticlockwise rotation. Moreover, well after the colonization took place, northward range expansion might have produced the presently observed genetic diversity, as inferred from haplotype and nucleotide diversity estimates. On the whole, this study highlights the evolution of Italian Reticulitermes taxa and supports the importance of a wide taxon sampling especially when dealing with organisms easily dispersed by human activities.

Phylogeography of two closely related species of Nolana from the coastal Atacama Desert of Chile: post‐glacial population expansions in response to climate fluctuations

AbstractAimTo investigate the impact of Quaternary climate cycles on the coastal Atacama Desert flora by assessing phylogeographical patterns of the desert shrub Nolana crassulifolia (Solanaceae) and its congener Nolana incana.LocationThe latitudinal aridity gradient from the southern margin of the coastal Atacama Desert to the mediterranean semi‐arid region of Chile (25–33° S).MethodsTwo cpDNA regions were sequenced for 130 individuals in 15 populations of these two closely related species, covering their entire distribution range (1000 km) along the arid to semi‐arid Chilean coast. We explored haplotype relationships in a statistical parsimony network, and assessed population genetic diversity, population differentiation and phylogeographical structure. In addition, we conducted demographic analyses and spatial analysis of genetic variation, identified barriers to gene flow with Monmonier's algorithm, and used Bayesian phylogenetic reconstruction to estimate divergence dates between lineages.ResultsWe found a total of 14 haplotypes – four of them shared by both species – and high levels of genetic differentiation among populations, but no past distribution breaks that could account for major vicariant events. Genetic diversity decreased continuously from north to south, with loss of haplotypes and a greater number of monomorphic populations in the southern range. Landscape analysis revealed greater genetic differentiation in the northernmost populations of both species.Main conclusionsThe documented north–south gradient of declining genetic diversity, the origin and location of ancestral haplotypes in northern sites, and the loss of haplotypes from southern populations all support the hypothesis of post‐glacial range expansion of Nolana southwards during arid/warmer cycles in the Atacama. The higher genetic diversity and greater differentiation of northern populations of both Nolana species support the hypothesis that populations survived in northern arid sites during wetter/colder episodes of the glacial cycles. We suggest that Quaternary arid phases in the Atacama promoted southward expansion of the coastal desert vegetation.

Mitochondrial DNA analyses reveal low genetic diversity in Culex quinquefasciatus from residential areas in Malaysia

The present study explored the intraspecific genetic diversity, dispersal patterns and phylogeographic relationships of Culex quinquefasciatus Say (Diptera: Culicidae) in Malaysia using reference data available in GenBank in order to reveal this species' phylogenetic relationships. A statistical parsimony network of 70 taxa aligned as 624 characters of the cytochrome c oxidase subunit I (COI) gene and 685 characters of the cytochrome c oxidase subunit II (COII) gene revealed three haplotypes (A1-A3) and four haplotypes (B1-B4), respectively. The concatenated sequences of both COI and COII genes with a total of 1309 characters revealed seven haplotypes (AB1-AB7). Analysis using tcs indicated that haplotype AB1 was the common ancestor and the most widespread haplotype in Malaysia. The genetic distance based on concatenated sequences of both COI and COII genes ranged from 0.00076 to 0.00229. Sequence alignment of Cx. quinquefasciatus from Malaysia and other countries revealed four haplotypes (AA1-AA4) by the COI gene and nine haplotypes (BB1-BB9) by the COII gene. Phylogenetic analyses demonstrated that Malaysian Cx. quinquefasciatus share the same genetic lineage as East African and Asian Cx. quinquefasciatus. This study has inferred the genetic lineages, dispersal patterns and hypothetical ancestral genotypes of Cx. quinquefasciatus.

Genetic Analysis and Captive Breeding Program Design for the Eastern Massasauga Sistrurus catenatus catenatus

Abstract The eastern massasauga Sistrurus catenatus catenatus is a declining species for which a captive breeding program was established in 2006. To effectively manage wild and captive populations, an understanding of genetic diversity within the species is necessary. We analyzed mitochondrial DNA sequences of 186 individuals: 109 wild snakes from 34 U.S. and Canadian counties and districts, all 52 breeding program members (23 of known and 29 of unknown origin), 18 other captives of unknown origin, and 7 outgroup representatives of desert massasauga S. c. edwardsii, and western massasauga, S. c. tergeminus. Statistical parsimony, maximum likelihood, and maximum parsimony analyses all identified eastern massasaugas as divergent from western and desert massasaugas. We found 18 different haplotypes among eastern massasaugas, comprising three geographically and genetically differentiated NADH dehydrogenase II (ND2) subunits that potentially reflect post-Pleistocene range expansion from unglaciated into formerly glaciated regions. Snakes of unknown origin could all be assigned unambiguously to these ND2 subunits. To maintain natural genetic variation, preserve diversity in captive lineages, and allow future augmentation or reintroduction, the Association of Zoos and Aquariums is managing these three geographic ND2 subunits separately within the Eastern Massasauga Species Survival Plan breeding program.

Molecular phylogenetics and phylogeographic structure of Sumichrast’s harvest mouse (Reithrodontomys sumichrasti: Cricetidae) based on mitochondrial and nuclear DNA sequences

Sumichrast’s harvest mouse (Reithrodontomys sumichrasti) is a montane rodent species widely distributed through the Mesoamerican highlands. We used sequence data from one mitochondrial (cytochrome b) and two nuclear (β-fibrinogen and acid phosphatase type V) genes for a total of 1962 base pairs to estimate genealogical relationships and assess population genetic structure across the range of this taxon. Maximum likelihood and Bayesian approaches using cytochrome b resolved several major clades, revealing considerably more genetic diversity than observed in previous studies. The basal split in the tree topologies corresponded to the geographical separation among samples on either side of the Isthmus of Tehuantepec in México. We estimated an early Pleistocene or late Pliocene divergence between these two groups. We also recovered a well-supported clade south of the Nicaraguan Depression in Central America that we consider a separate biological species. The 12 networks generated using statistical parsimony (TCS) for cytochrome b sequence data were largely concordant with the phylogenetic analyses and we document the co-occurrence of two of these networks in central Veracurz. Phylogenies derived from β-fibrinogen and acid phosphatase type V gene segments revealed less phylogenetic signal and did not separate samples of R. sumichrasti east and west of the Isthmus of Tehuantepec. The phylogeny estimated by combining the mitochondrial and nuclear sequence data was essentially identical to the cytochrome b gene tree.

Phylogeography of the neotropical Anopheles triannulatus complex (Diptera: Culicidae) supports deep structure and complex patterns.

BackgroundThe molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated.MethodsThe mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches.ResultsEach data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis.ConclusionCombination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.

DNA-barcoding contradicts morphology in quill mite species Torotrogla merulae and T. rubeculi (Prostigmata: Syringophilidae)

Torotrogla merulae Skoracki, Dabert et Ehrnsberger, 2000 and T. rubeculi Skoracki, 2004 have been considered as distinct steno- and monoxenous quill mite species (Acari: Prostigmata: Syringophilidae) parasitizing the thrushes of the genus Turdus Linnaeus and the European robin Erithacus rubecula (Linnaeus), respectively. Morphological and molecular studies on the taxonomical status of these two species provided contradictory results. Well defined differences in morphology were not supported by substantial genetic distance in nucleotide sequences of the DNA barcode (mitochondrial cytochrome c oxidase subunit I, COI, and D2 domain of the nuclear 28S rRNA gene), by the topology of the phylogenetic trees (neighbor-joining, maximum parsimony, maximum likelihood) and the network analyses of the COI haplotype genealogy (median-joining, statistical parsimony) that reveal rubeculi populations nested within merulae haplotypes. Since detected differences between T. merulae and T. rubeculi populations (1.6-2.4% for COI and 0.1% for D2) are comparable to the intraspecific level observed in majority of currently recognized European Torotrogla species and are much lower than the interspecific distances observed in the genus, we postulate their conspecificity. Because main morphological distinctions concern the structures used for feeding, we hypothesize that they are the result of phenotypic plasticity evoked by specific and different environmental conditions prevailing on the host bodies (thickness of the feather quill wall).

Molecular Taxonomy of Anopheles (Nyssorhynchus) benarrochi (Diptera: Culicidae) and Malaria Epidemiology in Southern Amazonian Peru

Anopheline specimens were collected in 2011 by human landing catch, Shannon and CDC traps from the malaria endemic localities of Santa Rosa and San Pedro in Madre de Dios Department, Peru. Most specimens were either Anopheles (Nyssorhynchus) benarrochi B or An. (Nys.) rangeli, confirmed by polymerase chain reaction-restriction fragment length polymorphism-internal transcribed spacer 2 (PCR-RFLP-ITS2) and, for selected individuals, ITS2 sequences. A few specimens from Lupuna, Loreto Department, northern Amazonian Peru, were also identified as An. benarrochi B. A statistical parsimony network using ITS2 sequences confirmed that all Peruvian An. benarrochi B analyzed were identical to those in GenBank from Putumayo, southern Colombia. Sequences of the mtDNA COI BOLD region of specimens from all three Peruvian localities were connected using a statistical parsimony network, although there were multiple mutation steps between northern and southern Peruvian sequences. A Bayesian inference of concatenated Peruvian sequences of ITS2 + COI detected a single clade with very high support for all An. benarrochi B except one individual from Lupuna that was excluded. No samples were positive for Plasmodium by CytB-PCR.

Precipitation rather than temperature influenced the phylogeography of the endemic shrub Anarthrophyllum desideratum in the Patagonian steppe

AbstractAimIn order to assess the impact of precipitation changes during Pleistocene glaciations on plant species of the Patagonian steppe, a phylogeographical study of the endemic shrub Anarthrophyllum desideratum was performed.LocationSouthern Patagonia: Argentina and Chile.MethodsChloroplast intergenic spacers trnS–trnG and rpoB–trnC were sequenced for 264 individuals from 33 localities spanning the entire distribution of A. desideratum. Phylogenetic (statistical parsimony, maximum likelihood and Bayesian inference) and population genetic analyses (spatial analyses of molecular variance, mismatch distributions, neutrality tests and Bayesian skyline plot) were performed. Divergence time estimates using a calibrated molecular clock were also conducted. Niche modelling was used to reconstruct the palaeodistribution to validate phylogeographical patterns.ResultsThirty haplotypes were identified that clustered into two main lineages, revealing a significant latitudinal phylogeographical break north and south of the Deseado River (c. 47° S). Infra‐specific diversification began in the late Miocene, with northern and southern lineages separating c. 3 Ma, after the eastern Patagonian lowlands started to become increasingly arid. Three areas of high molecular diversity were identified: one in southern and two in northern Patagonia where niche modelling indicates that the species may have survived during the Last Glacial Maximum. These putative refugia received more moisture than much of the steppe during glaciation‐associated aridization. The south‐western refugium is the more likely source for eastward range expansion during post‐glacial humidification.Main conclusionsAnarthrophyllum desideratum responded differently to historical processes north and south of the Deseado River. In the north this species survived in situ in fragmented populations, whereas in the south it survived in localized refugia that presumably avoided extreme aridization, and from which it expanded eastwards. For southern Patagonia, our results support a new historical scenario affected more by precipitation regimes than by temperature changes associated with glacial cycles. This hypothesis should be considered in future plant phylogeographical studies from the Patagonian steppe.