Complex Species Research Articles

Ecological overview of hard ticks (Ixodida: Ixodidae) in Nagasaki prefecture of western Japan during winter 2021–2022

In Japan, Japanese spotted fever, Lyme disease and severe fever with thrombocytopenia syndrome caused by Ixodidae species are endemic. To prevent and control the diseases, fundamental understandings in tick ecology are crucial. Hence, this study aimed to analyse tick species richness and abundance across Nagasaki prefecture including its remote islands from a wide range of environments. A total of 74 sampling points screening during winter 2021–2022, using dragging method resulted in 14,883 tick samples (279 adults, 7148 nymphs and 7456 larvae) in 11 species belonging to four genera. Haemaphysalis flava dominated adult populations, while H. formosensis was predominant among nymphs. Both species are possible vectors of Japanese spotted fever and severe fever with thrombocytopenia syndrome. The ecological analysis revealed more complex species distribution in the remote islands compering to the main island in both adults and nymphs. In addition, the ground temperature was a significant regulatory factor for both adults and nymphs. The research provides valuable insights on tick distributions, ecological groupings and environmental preferences in Nagasaki. These findings contribute to the fundamental understanding of tick ecology and could contribute to design strategies for tick population control and tick-borne disease prevention in Nagasaki or possibility to nearby areas.

A paradigm shift for evaluating natural attenuation of radioactive iodine in soils and sediments: Species-specific mechanisms and pathways.

The primary approach to assessing monitored natural attenuation (MNA) is currently based on a conceptual model utilizing the total contaminant concentrations, assuming a single aqueous species. However, many contaminants, such as metals and radionuclide - including iodine, can exist in multiple species that behave chemically differently in the environment and can exist simultaneously. For example, radioiodine often occurs concurrently as three major aqueous species: iodide (I-), iodate (IO3-), and organo-I, which undergo distinct attenuation pathways and exhibit markedly different mobility and geochemical behavior. Here, current literature is reviewed with the objective to: 1) demonstrate differences in iodine species' geochemical behavior and natural attenuation mechanisms; 2) show that a species-specific (or multi-species) approach provides greater details on contaminant migration and attenuation; and (3) discuss the logistics of a species-specific approach to developing conceptual models for assessing overall contaminant mobility. The species-specific approach results in a more accurate assessment of mass flux and maximum groundwater concentrations; and, therefore, a more defensible risk evaluation to support short- or long-term remediation and/or natural attenuation strategies. Although iodine is the focus of this paper, this methodology could be applied to other risk-driving contaminants such as mercury and uranium, which have even more complex aqueous speciation than iodine, or technetium and chromium, which have complex solid phase speciation and natural attenuation reaction networks. Accounting for species-specific geochemical behavior, while implementing MNA strategies can greatly reduce uncertainty, and, therefore, remedial costs required to ultimately achieve remediation regulatory objectives.

Morphological examination of Anthrenus oceanicus Fauvel (Coleoptera: Dermestidae: Megatominae)

A morphological examination of Anthrenus oceanicus Fauvel is presented. This species is considered a member of the Palaearctic Anthrenus pimpinellae species complex. Descriptions and images of habitus features, male terminalia, and sclerites within the bursa copulatrix are presented. The distribution of A. oceanicus is provided. Anthrenus oceanicus differs in many respects from all other members of the A. pimpinellae species complex. The conclusion is that A. oceanicus belongs to a different taxonomic lineage than the rest of the A. pimpinellae complex species.

Carbapenem-resistant Enterobacter hormaechei uses mucus metabolism to facilitate gastrointestinal colonization.

The emergence and global spread of carbapenem-resistant Enterobacter cloacae complex species present a pressing public health challenge. Carbapenem-resistant Enterobacter spp. cause a wide variety of infections, including septic shock fatalities in newborns and immunocompromised adults. The intestine may be a major reservoir for these resistant strains, either by facilitating contamination of fomites and transfer to susceptible individuals, or through translocation from the gut to the bloodstream. For this reason, we sought to establish a neonatal mouse model to investigate the mechanisms underpinning gut colonization by carbapenem-resistant Enterobacter hormaechei. We describe a new mouse model to study gut colonization by Enterobacter spp., leading to vital insights into the adaptation of carbapenem-resistant E. hormaechei to the gut environment during the early stages of intestinal colonization. We observed successful colonization and proliferation of E. hormaechei in the 5-day-old infant mouse gut, with primary localization to the colon following oral inoculation. We also uncovered evidence that E. hormaechei uses mucus as a carbon source during colonization of the colon. Our findings underscore the importance of oxygen-dependent metabolic pathways, including the pyruvate dehydrogenase complex and N-acetyl-D-glucosamine metabolism, in gut colonization and proliferation, which aligns with previous human studies. These insights are essential for developing novel therapeutic strategies that can serve as decolonization therapies in at-risk populations.IMPORTANCEBloodstream infections caused by Enterobacter spp. pose a significant clinical threat. The intestine acts as the primary site for colonization and serves as a reservoir for infection. To combat this pathogen, it is crucial to understand how carbapenem-resistant Enterobacter spp. colonize the gut, as such knowledge can pave the way for alternative therapeutic targets. In this study, we developed a novel neonatal mouse model for gastrointestinal colonization by Enterobacter spp. and discovered that mucus plays a key role as a carbon source during colonization. Additionally, we identified two mucus catabolism pathways that contribute to intestinal colonization by carbapenem-resistant E. hormaechei. This new mouse model offers valuable insights into host-pathogen interactions and helps identify critical gastrointestinal fitness factors of Enterobacter, potentially guiding the development of vaccines and alternative therapeutic strategies to minimize intestinal carriage in patient populations at risk of infection with Enterobacter spp.

SiO2-Mediated Hydrothermal Synthesis of Spiroffite-Type Co2Te3O8.

The hydrothermal synthesis of novel materials typically relies on both knowledge of the redox activities of all cations present in the reaction solution and a small toolset of so-called mineralizers to tune the solution's overall chemical potential. Upon the use of a less conventional mineralizer species, SiO2, we show the stabilization of spiroffite-type Co2Te3O8 under less forceful hydrothermal conditions than those in previous reports. When synthesized in the presence of both SiO2 and each respective alkali carbonate as a secondary mineralizer, silicon substitution in place of tellurium in the host structure becomes apparent, and the corresponding disorder introduced gives rise to enhanced low-temperature ferromagnetism. Our results highlight the complexities of underutilized and combined mineralizer species in the stabilization and tuning of complex magnetic ground states via hydrothermal synthesis techniques.

Nanoscale Probing of the Organic Binder in Artists' Paint Layers: Organic Phases and Chemical Heterogeneity.

Understanding paint structures at the nanoscopic level can address key questions related to artistic techniques, paint formulation, and long-term preservation of artworks. This involves examining spatial chemical complexity, the formation of molecular networks, and interactions between organic and inorganic constituents. Depending on the paint preparation methods, proteins and drying oils, the most common binders in traditional artistic practices, can be integrated to produce paints with diverse structures and nanoscale chemical intricacies. In this study, we utilize atomic force microscopy-based infrared spectroscopy (AFM-IR) to investigate the spatial chemical complexity and reaction pathways of organic species in artists' paints, including oil, tempera, and mixed-media tempera grassa. By analyzing these paints at the nanoscale, we established connections between their structural organization, chemistry, and formulation.

Silver N-heterocyclic biscarbene complexes: Potent inhibitors of thioredoxin reductase with anticancer activity in vitro and in vivo.

Silver N-heterocyclic carbene (NHC) complexes are known to form biscarbene species from monocarbene analogs in protic polar solvents. However, the effect of the respective species of silver NHC complexes on their biological activity against bacteria or cancer cells has not been systematically explored, either in vitro or in vivo. The direct and simple conversion of monocarbene silver N-heterocyclic carbene (NHC) halide complexes (NHC)AgX, (X= Cl, Br) 1a/b - 5a/b to their biscarbene analogues (NHC)2AgX 1c/d - 5c/d is reported. The biscarbenes demonstrated generally lower activity against bacteria compared to the monocarbene complexes; however, both types showed similar activity against tumor cells and a non-tumor reference cell line. Selected mono- and biscarbene complexes 3a and 3c showed similar strong inhibitory effects on thioredoxin reductase in vitro and in cellulo and had a similar level of metal uptake into A549 cells. The subsequent evaluation of their effects in vivo revealed relatively low toxicity and high antitumoral efficacy of both selected complexes in mice. The biscarbene silver organometallic 3c showed the most pronounced reduction of tumor growth in animals. The results indicate that both (NHC)AgX and (NHC)2AgX complexes trigger anticancer activity as biscarbene complexes, making this the preferred form for future metallodrug development.

Presence of the Anopheles culicifacies complex species A in southeast Iran

BackgroundThe Anopheles culicifacies complex is one of the most important malaria vectors in Southeast Asia and Southeastern Iran. Although the sibling species within this complex are morphologically indistinguishable, they differ significantly in their disease transmission potential, blood-feeding behaviour, and other biological traits. Cytogenetic and chromosomal studies have identified five sibling species within this complex: A, B, C, D, and E. Understanding the species composition and distribution of this complex is crucial for malaria control strategies.ObjectivesThis study aimed to identify the sibling species of the An. culicifacies complex in Qaleh Ganj County, Kerman Province, Southeastern Iran. Specifically, the study sought to determine the presence of species A, which is known to be a primary vector of malaria in the region.MethodsWe employed allele-specific PCR and sequencing PCR techniques to identify the sibling species. DNA was extracted from mosquito specimens, and the D3 region of the 28S rDNA gene and a segment of the COII gene from the mitochondrial genome (mtDNA) were targeted for amplification and sequencing.ResultsData analysis revealed a positive correlation between An. culicifacies s.l. specimens and altitude, with most specimens collected from mountainous areas. Both allele-specific PCR and sequencing PCR confirmed the presence of species A in the study areas of Kerman Province.ConclusionsGiven that species A is a primary malaria vector, the findings of this study provide valuable insights for guiding malaria control strategies in Southeastern Iran. Further studies are recommended to assess the vector competence and ecological dynamics of other species within the An. culicifacies complex in the region.

Potential of Ionizing Radiation Application for Reducing Environmental Risks Related to Solids Residues

The enormous amount of solids residues and related cost for a suitable disposal have increased in megacities as São Paulo and Buenos Aires. Inovative technologies may be used in order to face managing progams with inovative techniques for for recycling or recover of materials. Gamma radiation was applied in laboratory scale for two different types of residues: fungi control in vehicular air filters, and for rat bedding waste (RBW) disinfection. Both materials may be used after irradiation and the radiation doses required will be discussed. Part of experiments carried out in Argentina evidenced the suitable use of vermicomposting for growing Calendula officinalis. Radiation doses from 10 kGy up to 30 kGy were tested for controlling microorganisms during the projects and were very effective. For filters disinfection experiments, DNA sequencing analysis for the fungi that survived after irradiation not only confirmed the presence of Penicillium, Fusarium and Aspergillus (toxigenic), fungi but also improved the data: the contamination included Aspergillus niger, Aspergillus flavus, Penicillium glabrum specie complex and Fusarium incarnatum-equiseti specie complex.

Complex species structure of Lecithaster salmonis (Digenea: Lecithasteridae), a fish parasite in the Arctic and Pacific Northwest.

Studying complexes of cryptic or pseudocryptic species opens new horizons for the understanding of speciation processes, an important yet vague issue for the digeneans. We investigated a hemiuroidean trematode Lecithaster salmonis across a wide geographic range including the northern European seas (White, Barents, and Pechora), East Siberian Sea, and the Pacific Northwest (Sea of Okhotsk and Sea of Japan). The goals were to explore the genetic diversity within L. salmonis through mitochondrial (cox1 and nad5 genes) and ribosomal (ITS1, ITS2, 28S rDNA) marker sequences, to study morphometry of maritae, and to revise the life cycle data. Mitochondrial markers showed that L. salmonis is likely divided into six lineages (referred to as operational taxonomic units, OTUs), which often occur in sympatry, sometimes in a single host specimen. Variation in rDNA was not consistent with that in the mitochondrial markers. Morphometric analysis of maritae was performed for four out of six OTUs; it showed that some OTUs had significant differences from the others, but some did not. The effect of host species on the morphometric characteristics cannot be excluded. Intramolluscan stages were identified for two OTUs; they differed clearly by the structure of cercariae and also by the species of the first intermediate host. The case of L. salmonis is instructive in how different criteria for species delimitation can contradict each other. We regard this as a sign of recent or ongoing speciation and suggest using the name Lecithaster cf. salmonis. The most promising criteria to differentiate genetic lineages within L. cf. salmonis are first intermediate hosts and morphological characteristics of the cercariae: shape of the delivery tube and caudal cyst, and length of the filamentous appendage.

Spectroscopic Study on Identifying Synergistic Extraction Complexes of Nd(III) with Bis(2,4,4-Trimethylpentyl)Dithiophosphinic Acids and TBP

ABSTRACT Selective separation of trivalent actinide ions, An(III), from trivalent actinide ions, Ln(III), has been of great importance and considerable challenge in terms of the overall nuclear fuel cycle strategy. The construction of the hard-soft synergistic extraction system, commonly S containing-O containing ligands, aims to achieve the effective separation of the two metal ions. Nevertheless, the precise mechanism by which this system extracts metal ions remains the subject of controversy. Herein, the complexation of Nd(III) with bis(2,4,4-trimethylpentyl)dithiophosphinic acid (HL) and tributyl phosphate (TBP) has been investigated with spectroscopy and extraction experiments under different conditions to clarify the mechanisms in synergistic extraction. Five complex species, NdL3(HL)(TBP), NdL2(NO3)(TBP)3, NdL3(H2O)2(TBP), NdL3(H2O)(TBP)2, and NdL3(TBP)3, are identified in the synergistic extraction system, their molar absorption spectra are deconvoluted, and the corresponding extraction equilibrium constants are determined. In addition, a complex species of Nd(III) fully coordinated by TBP, NdL3(TBP)n (n ≥ 6), is found in titrations of organic solution containing NdL3(TBP)3 with more TBP, but it is not found in synergistic extraction. Furthermore, Density Functional Theory (DFT) is employed to confirm the stability of the complexes. Investigation of the mechanism of this system will facilitate the acquisition of further insights into the selection of O-containing ligands and the development of synergistic extraction process for the separation of An(III) and Ln(III).

Whole-genome phylogenetic analysis of Mycobacterium avium complex from clinical respiratory samples.

Mycobacterium avium complex (MAC) infections are increasingly challenging to manage due to their complex species diversity and varied resistance patterns. This study underscores the genetic diversity within MAC, identifying at least eight species and subspecies among 203 clinical isolates, with M. avium subsp. hominissuis (MAH) being most prevalent at 72.9%. Notably, genetic clustering was observed within MAH and M. intracellulare subsp. chimaera, suggesting potential transmission routes within healthcare settings. Clarithromycin and amikacin resistance was found to be uncommon, aligning with the rarity of resistance-associated genetic mutations. These findings emphasize the need for enhanced infection control measures and routine susceptibility testing to tailor antibiotic therapies effectively.

Hybridization and diversity of the genus Vandenboschia in Korea insights from morphological, cytological, and genotype analyses

The genus Vandenboschia exhibits significant diversity, driven by complex hybridization events and varying ploidy levels in the natural habitats of Korea, leads to misidentifications that obscure its true distribution and classification. To address this issue, the present study employed morphological, cytological, and genotype analyses to clarify the taxonomical circumscription and distribution patterns of the Vandenboschia radicans complex in Korea. The V. radicans complex in the genus refers to V. radicans and its closely related species and exhibits significant morphological variation, making classification challenging. Previous molecular studies have revealed that it consists of several non-hybrid and hybrid species among them. Through the present study, comprehensive sampling from Jeju and Ulleung Islands identified five distinct taxa from the V. radicans complex: two non-hybrid species (Vandenboschia kalamocarpa and Vandenboschia nipponica), and three hybrids (Vandenboschia × stenosiphon, Vandenboschia × quelpaertensis, and Vandenboschia kalamocarpa × Vandenboschia nipponica × Vandenboschia striata). Notably, hybrids exhibited broader geographic distributions compared to non-hybrid species, which were confined to specific microhabitats. While overlapping morphological traits among the species complicate identification without genetic analysis, rhizome diameter and the width-to-length ratio of the involucre can serve as key morphological traits for distinguishing members of the Vandenboschia radicans complex in Korea. Additionally, cleaved amplified polymorphic sequence (CAPS) markers and cloning techniques were employed for species identification, particularly when morphological traits were inconclusive. These findings highlight the need to integrate genetic tools with traditional taxonomic methods to resolve complex species relationships within Vandenboschia in Korea and emphasize the importance of conservation efforts for geographically restricted and genetically distinct populations.

The planktonic freshwater ciliate Balanion planctonicum (Ciliophora, Prostomatea): A cryptic species complex or a "complex species"?

The globally distributed ciliate Balanion planctonicum is a primary consumer of phytoplankton spring blooms. Due to its small size (~20 μm), identification and quantification by molecular tools is preferable as an alternative to the laborious counting of specimen in quantitative protargol stains. However, previous sequencing of the 18S rDNA V9 region of B. planctonicum from Lake Zurich (Switzerland) and subsequent quantification by fluorescence insitu hybridization yielded significantly lower cell numbers than using morphotype counting. This raised the question of whether B. planctonicum shows a cryptic diversity or whether it is just a 'complex species' with intra-clonal polymorphisms. Over three years, we established numerous monoclonal cultures, and long-read sequencing of rDNA operons revealed four distinct dominant haplotypes (BpHs 1-4). The gene sequences of BpHs 1 and 3 differed by 6% and did not share intra-clonal polymorphisms, providing evidence for two distinct clades. Furthermore, phylogenetic analyses corroborate the sister relationship between Balanion and Askenasia (plus Hexasterias and Radiosperma). Morphologically, the two Balanion clades are nearly indistinguishable with small differences in macronucleus size and in the cell length to width ratio. CARD-FISH analyses indicated that the diversity of B. planctonicum is even more extensive with still unidentified clades.

Incidence of mycobacteria in pulmonary granulomatous lesions.

Mycobacteria infections are caused by species of the Mycobacterium tuberculosis complex (MTB) and other species called Non-Tuberculosis Mycobacteria (NTM). Identification of mycobacteria species is very important to define treatment and it can be achieved by direct culture. However, the lack of clear protocols regarding the use of culture or molecular tests on specimens diagnosed with granulomatous lesions causes delays in the diagnosis of the etiological agents and, consequently, the definition of the right treatment. This work aimed to characterize the incidence of mycobacteria species in pulmonary granulomatous lesions and the contribution of Polymerase Chain Reaction (PCR) in Formalin-Fixed Paraffin-Embedded Tissue (FFPE), direct culture, and Ziehl-Neelsen histological stain to the diagnosis. The authors performed an observational, centralized, and retrospective study in a cohort of 336 cases with pulmonary granulomatous lesions. Mycobacteria were detected by ZNS in 54/323 (16.72 %) and by direct culture in 40/198 (20.20 %). MTB DNA was detected by PCR in 10/57 (17.54 %). Mycobacterial culture results revealed MTB in 26/40 (65.00 %), whereas NTM was detected in 13/40 (32.50 %). NTM was represented by M. avium (n = 4), M. intracellulare (n = 3), M. kansasii (n = 3), M. colombiense (n = 1), M. paraffinicum (n = 1), and M. abscessus subsp. massiliense (n = 1). In conclusion, this study demonstrated that mycobacteria are detected in 16.72 % to 20.20 % of pulmonary granulomatous lesions. Moreover, MTB and NTM were detected in these lesions. The use of different methods for mycobacteria detection, in addition to culture, is complementary and contributes to fastening and increasing the detection of mycobacteria in these lesions.

Aqueous Synthesis of Membrane Lipids via Amide Formation between Amphiphilic Amines and Thioacids

Protocell membranes were likely formed through primitive metabolic reactions that synthesized more complex membrane lipid species from simpler precursors. Thioacids have been shown to be prebiotically plausible and chemoselective N‐acylating reagents. They have been successfully employed for the acylation of peptides and RNA. However, the ability of thioacids to acylate lipid species has not yet been explored. Here, we demonstrate N‐acylation of natural amine‐containing amphiphiles using fatty thioacids. This reaction occurs spontaneously in aqueous conditions, is chemoselective, and traceless. The synthesis of several natural sphingolipids via direct N‐acylation is possible and their subsequent de novo formation into vesicular membranes is shown. Aqueous thioacid coupling provides a route for generating membrane‐forming lipids in the context of protocells and for the in situ synthesis of relevant sphingolipid species.

Genetic Relationships and Molecular Signatures of Divergence in Traditional Landraces and Morphotypes of Brassica oleracea.

Brassica oleracea is a complex species incorporating a great variety of vegetable types, including cabbage, cauliflower, broccoli, kale, and others. Southern Italy, and especially the Puglia region, is rich in B. oleracea landraces. In this study, genotyping-by-sequencing (GBS) was applied to a germplasm panel of 82 samples, mostly landraces and some commercial varieties, belonging to various morphotypes of B. oleracea. Population structure (K = 2), principal component (PCA), and phylogenetic analyses resulted in a general subdivision of our samples into two large lineages: the types used for their leaves (LHL) and those consumed for their flower heads (AIL). Going deeper inside, the different morphotypes were mostly grouped into specific clusters, and a clear separation of particular landraces, such as the Mugnoli and Cima nera broccoli, was observed in the structure analysis (K = 7), as well as in the PCA and in the Neighbor-Joining tree. The calculation of the pairwise fixation index (FST, threshold > 0.50) between LHL and AIL types (based on population structure analysis at K = 2) provided 456 outlier single nucleotide polymorphisms (SNPs). Among the corresponding orthologs annotated in Arabidopsis, we identified several genes involved in flower/inflorescence development, cellular proliferation, etc. Overall, our investigation provides useful information on the knowledge of early domesticated landraces of B. oleracea and allows for the attribution of unknown material to the appropriate taxonomical ranking. The analysis of outlier SNPs has highlighted signatures of molecular divergence between LHL and AIL lineages.

Mutual neutralization of C60+ and C60− ions

Context. Mutual neutralization (MN) between cations and anions plays an important role in determining the charge balance in certain astrophysical environments. However, empirical data for such reactions involving complex molecular species have been lacking due to challenges in performing experimental studies, leaving the astronomical community to rely on decades-old models with large uncertainties for describing these processes in the interstellar medium. Aims. Our aim is to investigate the MN reaction C60+ + C60− → C60* + C60 for collisions at interstellar-like conditions. Methods. We studied the MN reaction between C60+ and C60− at collision energies of 100 meV using the Double ElectroStatic Ion Ring ExpEriment (DESIREE) and its merged beam capabilities. To aid in the interpretation of the experimental results, semiclassical modeling based on the Landau-Zener approach was performed for the studied reaction. Results. We experimentally identified a narrow range of kinetic energies for the neutral reaction products. Modeling was used to calculate the quantum state-selective reaction probabilities, absolute cross sections, and rate coefficients of these MN reactions, using the experimental results as a benchmark. We compared the MN cross sections with model results for electron attachment to C60 and electron recombination with C60+. Conclusions. Our results show that it is crucial to take mutual polarization effects, the finite sizes, and the final quantum states of both molecular ions into account in order to obtain reliable predictions of MN rates expected to strongly influence the charge balance and chemistry in environments such as dense molecular clouds.

Extensive genome-wide phylogenetic discordance is due to incomplete lineage sorting in the rapidly radiated East Asian genus Nekemias (Vitaceae)

Abstract Background and Aims Nekemias is a small genus of the grape family, with nine species discontinuously distributed in temperate to subtropical zones of the Northern Hemisphere but mostly in East Asia. Previous phylogenetic studies on Nekemias have mainly been based on a few chloroplast markers, and the phylogenetic framework and systematic relationships are still highly contested. Methods We carried out a systematic framework reconstruction of Nekemias and intra-generic reticulate evolutionary analyses based on extensive single-copy nuclear and chloroplast genomic data obtained by the Hyb-Seq approach, combining genome skimming and target enrichment. Key Results Both nuclear and chloroplast genomic data strongly support the monophyly of Nekemias with its division into two major lineages from East Asia and North America, respectively. There are strong and extensive topological conflicts among nuclear gene trees and between nuclear and chloroplast topologies within the genus, especially within the East Asian clade. Conclusions Rapid radiation through predominant incomplete lineage sorting (ILS) throughout the evolutionary history of the East Asian taxa is supported to explain the relatively high species diversity of Nekemias in East Asia. This study highlights the important role of short periods of rapid evolutionary radiations accompanied by ILS as a mechanism for the complex and fast species diversifications in the grape family as well as potentially in many other plant lineages in East Asia and beyond.

Extended phylogenetic analysis and morphology of the Iresine cassiniiformis – I. hartmanii alliance (Amaranthaceae) reveals two further geographically distinct species

Background: Shrubs recognized as I. hartmanii were known from the northern Mexican deserts, whereas I. cassiniiformis is considered to occur throughout the Mexican highlands. Morphologically similar individuals were reported under both names also south beyond the Isthmus of Tehuantepec to Guatemala. Question: Are I. hartmanii and I. cassiniiformis distinct species, or do they represent widespread taxa exhibiting clinal variation due to adaptation along a gradient spanning desert to evergreen tropical forest? Taxon: Amaranthaceae, Iresine. Study site: Mexico and Central America. Method: Field and herbarium work, molecular phylogenetics, morphology, palynology, distribution mapping. Results: Plastid and nuclear phylogenetic trees suggest a complex speciation scenario in Iresine, revealing new lineages in the mountains of Chiapas, Oaxaca and Guatemala that also differ by morphological characters. Iresine cassiniiformis appears monophyletic and occurs in the Trans-Mexican Volcanic Belt whereas I. hartmanii comprises a core clade of individuals confined to the northern deserts. Specimens morphologically intermediate to I. cassiniiformis, and part of the I. hartmanii species clade in nrITS, occur directly south of the desert zone, either representing ancestral types in the speciation of I. hartmanii or introgression from I. cassiniiformis. Conclusion: Our results underscore the importance of an integrative taxonomy approach including molecular phylogenetics and morphology with a dense sampling of putative species from throughout the geographical range in order to arrive at explicit hypotheses on species limits. Here this supports the description and naming of two new species.