Cultivation strategies at the ancient Luanzagangzi settlement on the easternmost Eurasian steppe during the late Bronze Age

A significant body of recent research shows that the first east–west transmission of cereal crops, Triticum spp. (wheat) and Hordeum spp. (barley) from the west and millets (Setaria italica, foxtail millet, and Panicum miliaceum, common millet) from the east, took place sometime around the start of the 5th millennium bp, with part of the most likely route lying along the Tianshan mountains in northern Xinjiang, China. Here the dominant economic adaptation is, and was in prehistory, not crop-based agriculture but transhumant pastoralism. The site of Luanzagangzi (ca. 3,300–2,900 cal bp) on the northern slope of the Tianshan is one of only a handful of Bronze Age sites in Xinjiang with evidence for well-established crop cultivation. In this paper, we report on ten samples collected for phytolith analysis from a 4 m deep profile at Luanzagangzi. The results show evidence that a range of cereal crops was being grown (multi-cropping), Triticum spp., Hordeum spp., Setaria italica and Panicum miliaceum. Pooideae, Paniceae woody plants, Phragmites (reed) and Cyperaceae (sedges) were presumably also exploited for subsistence purposes in this area. We speculate that the strategy of growing a range of crops, wheat/barley, common millet and foxtail millet was adopted by the Bronze Age population in this region as a supplement to herding. The findings of this study help us to understand the dispersal of cultivation strategies across the Eurasian steppe via the Xinjiang region, and the communication between China and the West in the late Bronze Age.     

Selection and evaluation of reference genes for quantitative gene expression analysis in broomcorn millet (<Emphasis Type="Italic">Panicum miliaceum</Emphasis> L.)

The appropriate reference genes are crucial for normalization of the target gene expression in qRT-PCR analysis. Broomcorn millet (Panicum miliaceum L.) is one of the most important crops in drought areas worldwide, while the systematical investigation and evaluation of reference genes has not been investigated in this species up to now. Here, 9 commonly used reference genes were selected to detect their expressional stability in different tissues and under different stresses in broomcorn millet. ΔC_t, BestKeeper, NormFinder and GeNorm approaches were used to evaluate the potentiality of these candidate genes as the reference gene in broomcorn millet. Taken together, results found that 18S and GAPDH were the suitable reference genes for gene expression normalization in different tissues and under stress treatment in broomcorn millet. This was the first study to investigate the reference genes for qRT-PCR analysis in broomcorn millet, which will facilitate the gene expression studies and also accelerate revealing the molecular mechanism of well-adapted extreme climatic conditions.     

Screening of rice landraces (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for seedling stage salinity tolerance using morpho-physiological and molecular markers

Traditional rice landraces of coastal area in Bangladesh are distinct regarding their phenotype, response to salt stress and yield attributes. With characterization of these landraces, suitable candidate genes for salinity tolerance could be identified to introgress into modern rice varieties. Therefore, the aim of this experiment was to uncover prospective rice landraces tolerant to salinity. Relying on morphological, biochemical and molecular parameters 25 rice genotypes were tested for salt tolerance at germination and seedling stage. At germination stage 0 and 12 dSm^?1 salinity were imposed on rice genotypes. Ward’s cluster analysis divided rice genotypes into three clusters (susceptible, moderately tolerant and tolerant) based on the physiological indices. The tolerant rice landraces to salinity were Sona Toly, Nakraji and Komol Bhog. At seedling stage screening was performed following IRRI standard protocol at 12 dSm^?1 salinity level. Based on all morphological and biochemical parameters Komol Bhog was identified as the highly salinity tolerant landrace while Bolonga, Sona Toly, Dud Sail, Tal Mugur and Nakraji were found as tolerant to salinity. Molecular characterization using two simple sequence repeats (SSR) markers, viz. RM121 and RM337 displayed Bolonga, Til Kapor, Panbra, Sona Toly, Bina Sail, Komol Bhog, Nakraji, Tilkapur, Gajor Goria and Gota were tolerant landraces through genetic similarity in dendrogram. These identified salt-resistant landraces can be used as promising germplasm resources for breeding salt-tolerant high-yielding rice varieties in future.     

Geometric morphometric analysis of <Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv. (foxtail millet) and <Emphasis Type="Italic">Brachiaria ramosa</Emphasis> (L.) Stapf. (browntop millet) and its implications for understanding the biogeography of small millets

Setaria italica (L.) P. Beauv. (foxtail millet) was originally domesticated in northern China. The time and route of its introduction into South Asia is currently unclear due to the possible confusion with autochthonous Brachiaria ramosa (L.) Stapf. (browntop millet). Geometric morphometrics (GM) offer an alternative to traditional archaeobotanical methods to distinguish between these two small millet species. This study aims at finding a method to securely distinguish among charred caryopses of S. italica and B. ramosa, testing its validity on archaeobotanical assemblages and proposing a new approach for studying the dispersion of S. italica throughout Eurasia. Modern S. italica (n = 35) and B. ramosa (n = 34) caryopses and 15 archaeological specimens from a 5th millennium bp archaeological occupation site in northwestern India were analysed. Archaeological and modern caryopses (before and after charring) were photographed with a Leica EZ4D stereoscope, and TPSdig software was used to scale the photographs and manually apply a configuration of three landmarks and six semi-landmarks onto the contours of the embryos. Multivariate statistics were carried out to analyse the shape differences between modern S. italica and B. ramosa and to classify the archaeological specimens. The results show that the shape of the embryo of both species can be clearly distinguished using a GM-approach, both before and after charring. However, charring tends to smooth the shape differences between the two groups, which may affect the interpretation of archaeobotanical assemblages. The comparison between modern and archaeological caryopses suggests that S. italica was not present in northwestern India during the 5th millennium bp.     

High-throughput sequencing of <Emphasis Type="Italic">Prunus ferganensis</Emphasis> indicates that it is a geographical population of <Emphasis Type="Italic">P. persica</Emphasis>

Peach belongs to the genus Prunus, which includes Prunus persica and its relative species, P. mira, P. davidiana, P. kansuensis, and P. ferganensis. Of these, P. ferganensis have been classified as a species, subspecies, or geographical population of P. persica. To explore the genetic difference between P. ferganensis and P. persica, high-throughput sequencing was used in different peach accessions belonging to different species. First, low-depth sequencing data of peach accessions belonging to four categories revealed that similarity between P. ferganensis and P. persica was similar to that between P. persica accessions from different geographical populations. Then, to further detect the genomic variation in P. ferganensis, the P. ferganensis accession “Xinjiang Pan Tao 1” and the P. persica accession “Xia Miao 1” were sequenced with high depth, and sequence reads were assembled. The results showed that the collinearity of “Xinjiang Pan Tao 1” with the reference genome “Lovell” was higher than that of “Xia Miao 1” and “Lovell” peach. Additionally, the number of genetic variants, including single nucleotide polymorphisms (SNPs), structural variations (SVs), and the specific genes annotated from unmapped sequence in “Xia Miao 1” was higher than that in “Xinjiang Pan Tao 1” peach. The data showed that there was a close distance between “Xinjiang Pan Tao 1” (P. ferganensis) and reference genome which belong to P. persica, comparing “Xia Miao 1” (P. persica) and reference ones. The results accompany with phylogenetic tree and structure analysis confirmed that P. ferganensis should be considered as a geographic population of P. persica rather than a subspecies or a distinct species. Furthermore, gene ontology analysis was performed using the gene comprising large-effect variation to understand the phenotypic difference between two accessions. The result revealed that the pathways of gene function affected by SVs but SNPs and insertion-deletions markedly differed between the two peach accessions.     

Molecular characteristics of two new <Emphasis Type="Italic">waxy</Emphasis> mutations in China waxy maize

The waxy gene mutation causes waxy maize grain to have a sticky quality. China has numerous waxy maize landraces and is thought to be the place of origin of waxy maize. The most abundant waxy maize resources in China are located in the Yunnan province and its surrounding areas. We collected 57 waxy maize landraces from Yunnan province and cloned and sequenced the waxy gene from its fourth to eighth exon. Two new waxy gene mutations, named wx-Cin4 and wx-124, were identified. The wx-Cin4 mutation is a 466-bp retrotransposon inserted into exon six. The wx-124 mutation is a 116-bp miniature inverted-repeat transposable element inserted into exon seven. This is the first time a 124-type mutation has been found in a maize waxy gene. The discovery of the two specific waxy mutations from landraces collected in Yunnan province provides new evidence supporting the hypothesis that China is the origin area for waxy maize.     

Physiological and biochemical parameters: new tools to screen barley root exudate allelopathic potential (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L. subsp. <Emphasis Type="Italic">vulgare</Emphasis>)

Morphological markers/traits are often used in the detection of allelopathic stress, but optical signals including chlorophyll a fluorescence emission could be useful in developing new screening techniques. In this context, the allelopathic effect of barley (Hordeum vulgare subsp. vulgare) root exudates (three modern varieties and three landraces) were assessed on the morphological (root and shoot length, biomass accumulation), physiological (F_v/F_m and F₀), and biochemical (chlorophyll and protein contents) variables of great brome (Bromus diandrus Roth., syn. Bromus rigidus Roth. subsp. gussonii Parl.). All the measured traits were affected when great brome was grown in a soil substrate in which barley plants had previously developed for 30 days before being removed. The response of receiver plants was affected by treatment with activated charcoal, dependent on barley genotype and on the nature of the growing substrate. The inhibitory effect was lower with the addition of the activated charcoal suggesting the release of putative allelochemicals from barley roots into the soil. The barley landraces were more toxic than modern varieties and their effect was more pronounced in sandy substrate than in silty clay sand substrate. In our investigation, the chlorophyll content and F_v/F_m were the most correlated variables with barley allelopathic potential. These two parameters might be considered as effective tools to quantify susceptibility to allelochemical inhibitors in higher plants.     

Overview of the origins and history of the five major cucurbit crops: issues for ancient DNA analysis of archaeological specimens

The gourd family, Cucurbitaceae, contains five vegetable crops of worldwide importance, the pumpkins and squash (Cucurbita spp.), watermelons (Citrullus lanatus), melons (Cucumis melo) and cucumbers (Cucumis sativus). Here is presented a synopsis of the origin and history of these cucurbit crops. Historical records of the use of cucurbits by people take the form of archaeobotanical finds, iconography and literature. The weight of the evidence indicates that Cucurbita spp. were first cultivated in the Americas at least 10,000 years ago, and that by 1492 ce a number of cultivar-groups of pumpkins and squash had been developed by indigenous American peoples. Watermelons were cultivated in northeastern Africa at least 4,000 years ago, first probably as a source of fresh water. Melons and cucumbers are native to Asia, probably initially cultivated for the use of the young fruits as vegetables. Melons spread to eastern Africa at an early date, but cucumbers are probably a more recent domesticate and spread westwards later, reaching Europe in early medieval times. Sequencing of cucurbit plant genomes and advances in ancient DNA research offer much promise for obtaining an improved assessment of cucurbit crop origins, specifically the genetic constitution and geographical home of ancestral source populations. Next-generation genomic sequencing, if applied to an appropriate array of archaeological cucurbit remains and modern germplasm, could contribute much to the understanding of the history and evolution under domestication of cucurbit crops.     

The first record of <Emphasis Type="Italic">Pronotalia</Emphasis> Gradwell, 1957 (Hymenoptera,Eulophidae) Wasps from Georgia

Four species of Pronotalia Gradwell (Hymenoptera, Eulophidae: Tetrastichinae) are recorded from the Lagodekhi Reserve (Georgia). Formerly, only P. carlinarum (Szelényi et Erdös) was known from Transcaucasia (Armenia) (Kostjukov, 1978). Pronotalia fiorii (Domenichini), P. orobanchiae Graham, and P. trypetae Gradwell are recorded for Georgia and Transcaucasia for the first time. In addition to these species, only 4 species are known from Europe and Anatolia: P. erzurumica Doganlar, P. hungarica (Erdös), P. inflata Graham, and P. tortumensis Doganlar; thus, 50% of Pronotalia species from Europe and Anatolia occur in the Lagodekhi Reserve.     

Morphology and molecules reveal the alien <Emphasis Type="Italic">Posthodiplostomum centrarchi</Emphasis> Hoffman, 1958 as the third species of <Emphasis Type="Italic">Posthodiplostomum</Emphasis> Dubois, 1936 (Digenea: Diplostomidae) in Europe

Metacercariae of two species of Posthodiplostomum Dubois, 1936 (Digenea: Diplostomidae) were subjected to morphological and molecular studies: P. brevicaudatum (von Nordmann, 1832) from Gasterosteus aculeatus (L.) (Gasterosteiformes: Gasterosteidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Perca fluviatilis L. (Perciformes: Percidae), Czech Republic (morphology, cox1, ITS1-5.8S-ITS2 and 28S); and P. centrarchi Hoffman, 1958 from Lepomis gibbosus (L.) (Perciformes: Centrarchidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Slovakia (cox1 and ITS1-5.8S-ITS2). In addition, cercariae of P. cuticola (von Nordmann, 1832) from Planorbis planorbis (L.) (Mollusca: Planorbidae), Lithuania (morphology and cox1) and metacercariae of Ornithodiplostomum scardinii (Schulman in Dubinin, 1952) from Scardinius erythrophthalmus (L.) (Cypriniformes: Cyprinidae), Czech Republic, were examined (morphology, cox1, ITS1-5.8S-ITS2 and 28S). These represent the first molecular data for species of Posthodiplostomum and Ornithodiplostomum Dubois, 1936 from the Palaearctic. Phylogenetic analyses based on cox1 and ITS1-5.8S-ITS2, using O. scardinii as the outgroup and including the three newly-sequenced Posthodiplostomum spp. from Europe and eight published unidentified (presumably species-level) lineages of Posthodiplostomum from Canada confirmed the distinct status of the three European species (contrary to the generally accepted opinion that only P. brevicaudatum and P. cuticola occur in the Palaearctic). The subspecies Posthodiplostomum minimum centrarchi Hoffmann, 1958, originally described from North America, is elevated to the species level as Posthodiplostomum centrarchi Hoffman, 1958. The undescribed “Posthodiplostomum sp. 3” of Locke et al. (2010) from centrarchid fishes in Canada has identical sequences with the European isolates of P. centrarchi and is recognised as belonging to the same species. The latter parasite, occurring in the alien pumpkinseed sunfish Lepomis gibbosus in Europe, is also supposed to be alien for this continent. It is speculated that it colonised Europe long ago and is currently widespread (recorded in Bulgaria, Slovakia and Spain); based on the cox1 sequence of an adult digenean isolate from the Ebro Delta, Spain, only the grey heron (Ardea cinerea L.) (Ciconiiformes: Ardeidae) is known to be its definitive host in Europe.     

Genetic analysis of <Emphasis Type="Italic">NEKODE1</Emphasis> gene involved in panicle branching of foxtail millet, <Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv., and mapping by using QTL-seq

We carried out genetic analysis and mapping of a gene for the tip-branched panicle (Nekode or Neko-ashi in Japanese) in foxtail millet. We revealed that this trait is controlled by a single dominant gene by using two F₂ populations and designated the gene as NEKODE1. By using an F₂ population between closely related Taiwanese landraces with a new method based on next-generation sequencing (NGS), QTL-seq, we successfully and rapidly mapped the responsible gene (NEKODE1) on chromosome 9. We also mapped the gene by using SSR markers to verify that this gene is located at the position on chromosome 9, suggested by QTL-seq, and we obtained SSR markers closely linked to the gene and found several candidate genes for this trait in a foxtail millet genome sequence database. The use of a foxtail millet genome sequence and NGS enables rapid mapping of a gene(s) by using a segregation population derived from a cross even between closely related foxtail millet landraces.     

Hybridization success is largely limited to homoploid <Emphasis Type="Italic">Prunus</Emphasis> hybrids: a multidisciplinary approach

Prunus fruticosa is a rare shrub occurring in Eurasian thermophilous forest-steppe alliances. The species frequently hybridizes with cultivated Prunus species in Europe (allochthonous tetraploid P. cerasus and partly indigenous diploid P. avium). Propidium iodide flow cytometry, distance-based morphometrics, elliptic Fourier analysis and embryology were employed to evaluate the extent of hybridization in six Slovak populations. Flow cytometric analyses revealed three ploidy levels: diploid (P. avium), triploid (P. × mohacsyana) and tetraploid (P. fruticosa, P. × eminens and P. cerasus). In addition, P. fruticosa and P. cerasus, at the tetraploid level, were found to differ in absolute genome size. An embryological evaluation suggested the existence of a triploid block in P. × mohacsyana and significant potential for hybridization among tetraploid taxa (indicated also by a continuous distribution of genome size data and further mirrored by morphometrics). Although hybrids significantly differ in ploidy level and embryological characteristics, they are almost indistinguishable using morphological characters. Hybridization with P. cerasus thus turns out to be a significant threat to wild populations of P. fruticosa compared to the relatively weak influence of P. avium.     

NBS-LRR resistance genes polymorphism in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.) varieties inferred from NBS-profiling

NBS-profiling was used to study NBS-LRR resistance genes polymorphism in 32 Russian and foreign apple (M. domestica) varieties and nine Antonovka landraces. NBS-LRR resistance genes variability in the studied apple varieties was rather low. While in Antonovka landraces specific NBS-patterns were revealed, which may indicate the presence of a number of unique resistance genes. The results confirm that Antonovka landraces belong to M. domestica and Yakutskaya apple belongs to the section Gymnomeles.     

Phylogeny and morphology of the <Emphasis Type="Italic">Peziza ammophila</Emphasis> complex (Pezizales,Ascomycota), with description of two new species and a new form

This study was focused on species of Peziza belonging to the “P. ammophila” complex, using both morphological and molecular approaches. Molecular and morphological analyses showed that several taxa are hidden under the name “ammophila” and, as a consequence, two additional species and one form are established: P. hellenica sp. nov., P. oceanica sp. nov. and P. ammophila f. megaspora f. nov. On the basis of the data that we have, these taxa seem to be related to some geographical areas; P. ammophila appears to be prevalent in northern, central and southern Europe, its f. megaspora is distributed in France and in the Netherlands, P. hellenica comes from Greece and P. oceanica from New Zealand. A cryptic species, not yet circumscribed satisfactorily, is described as P. deceptiva ad int. Moreover, the problem concerning the identification of P. ammophila is raised, the species is epitypified with a sequenced collection from Sicily (Italy) and a key to the species of the complex is provided.     

Coding single nucleotide polymorphisms and SmCPS1 and SmKSL1 subcellular localization are associated with tanshinone biosynthesis in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bunge roots

Tanshinone is one of the major medicinal components of the roots of Salvia miltiorrhiza Bunge, and SmCPS1 and SmKSL1 are key enzymes in the tanshinone biosynthesis pathway. To increase our understanding of the coding single nucleotide polymorphisms (cSNPs) involved in tanshinone biosynthesis, seven S. miltiorrhiza landraces were examined. Our results revealed that the tanshinone content was significantly different among the seven landraces. In total, 48 cSNPs in SmCPS1 and 47 cSNPs in SmKSL1 were identified, and of these, 38 and 42 cSNPs, respectively, were associated with tanshinone content. The highest A/G and C/T base substitution rates were in SmCPS1 and SmKSL1, respectively. SmKSL1 expression was significantly, positively correlated with tanshinone IIA and tanshinone I contents, and SmCPS1 expression was significantly associated with tanshinone IIA content. Interestingly, subcellular SmCPS1 and SmKSL1 expression was enriched in the plastids. Therefore, cSNPs of SmCPS1 and SmKSL1 are involved in tanshinone biosynthesis in the plastids, where SmCPS1 and SmKSL1 enzymes catalyze tanshinone production in this species.     

Mycorrhization between <Emphasis Type="Italic">Cistus ladanifer</Emphasis> L. and <Emphasis Type="Italic">Boletus edulis</Emphasis> Bull is enhanced by the mycorrhiza helper bacteria <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Migula

Boletus edulis Bull. is one of the most economically and gastronomically valuable fungi worldwide. Sporocarp production normally occurs when symbiotically associated with a number of tree species in stands over 40 years old, but it has also been reported in 3-year-old Cistus ladanifer L. shrubs. Efforts toward the domestication of B. edulis have thus focused on successfully generating C. ladanifer seedlings associated with B. edulis under controlled conditions. Microorganisms have an important role mediating mycorrhizal symbiosis, such as some bacteria species which enhance mycorrhiza formation (mycorrhiza helper bacteria). Thus, in this study, we explored the effect that mycorrhiza helper bacteria have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this work was to optimize an in vitro protocol for the mycorrhizal synthesis of B. edulis with C. ladanifer by testing the effects of fungal culture time and coinoculation with the helper bacteria Pseudomonas fluorescens Migula. The results confirmed successful mycorrhizal synthesis between C. ladanifer and B. edulis. Coinoculation of B. edulis with P. fluorescens doubled within-plant mycorrhization levels although it did not result in an increased number of seedlings colonized with B. edulis mycorrhizae. B. edulis mycelium culture time also increased mycorrhization levels but not the presence of mycorrhizae. These findings bring us closer to controlled B. edulis sporocarp production in plantations.     

The seed dormancy allele <Emphasis Type="Italic">TaSdr</Emphasis>-<Emphasis Type="Italic">A1a</Emphasis> associated with pre-harvest sprouting tolerance is mainly present in Chinese wheat landraces

Key message

We cloned TaSdr - A1 gene, and developed a gene-specific marker for TaSdr - A1 . A QTL for germination index at the TaSdr - A1 locus was identified in the Yangxiaomai/Zhongyou 9507 RIL population.

Abstract

Pre-harvest sprouting (PHS) affects yield and end-use quality in bread wheat (Triticum aestivum L.). In the present study we found an association between the TaSdr-A1 gene and PHS tolerance in bread wheat. TaSdr-A1 on chromosome 2A was cloned using a homologous cloning approach. Sequence analysis of TaSdr-A1 revealed an SNP at position 643, with the G allele being present in genotypes with lower germination index (GI) values and A in those with higher GI. These alleles were designated as TaSdr-A1a and TaSdr-A1b, respectively. A cleaved amplified polymorphism sequence (CAPS) marker Sdr2A based on the SNP was developed, and linkage mapping and QTL analysis were conducted to confirm the association between TaSdr-A1 and seed dormancy. Sdr2A was located in a 2.9 cM interval between SSR markers Xgwm95 and Xgwm372. A QTL for GI at the TaSdr-A1 locus explained 6.6, 7.3, and 8.2 % of the phenotypic variances in a Yangxiaomai/Zhongyou 9507 RIL population grown at Beijing, Shijiazhuang, and the averaged data from the two environments, respectively. Two sets of Chinese wheat cultivars used for validating the TaSdr-A1 polymorphism and the corresponding gene-specific marker Sdr2A showed that TaSdr-A1 was significantly associated with GI. Among 29 accessions with TaSdr-A1a, 24 (82.8 %) were landraces, indicating the importance of Chinese wheat landraces as sources of PHS tolerance. This study identified a novel PHS resistance allele TaSdr-A1a mainly presented in Chinese landraces and it is likely to be the causal gene for QPhs.ccsu-2A.3, providing new information for an understanding of seed dormancy.

     

Adaptations to the hyporheic in Aloninae (Crustacea: Cladocera): allocation of <Emphasis Type="Italic">Alona protzi</Emphasis> Hartwig, 1900 and related species to <Emphasis Type="Italic">Phreatalona</Emphasis> gen. nov.

Morphological study of Alona protzi Hartwig, 1900, Alona phreatica Dumont, 1983 and Alona smirnovi Petkovski &; Flößner, 1972 reveals close affinities with Alona labrosa Vasiljeva &; Smirnov, 1969. We separate these four species from the polyphyletic Alona Baird, 1843 (Anomopoda: Chydoridae). United under Phreatalona gen. nov., these taxa share primitive features on the limbs, together with specializations to a rheic life mode. Phreatalona contains some of the only true hyporheic taxa within the Cladocera. Endemism in two ancient lakes (P. smirnovi and P. labrosa) and a preference for river sediments in Europe (P. phreatica and P. protzi) suggest a long isolation from typical littoral/benthic biotopes. We discuss close links with southern vicariant Nicsmirnovius, the position of these (hypo)rheic chydorids within the subfamily and their affinities with Acroperus. We remark an independent evolution of external (habitus, postabdomen) vs. internal (limb) morphology in the protzi-complex. Phreatalona is likely tertiary in origin, evolving from a littoral alonine adapting to rheic and finally hyporheic environments. Baikal endemic P. labrosa is likely the most primitive species of the genus. We discuss adaptations and evolution in the hyporheic and the effect on dispersal and biogeography of Phreatalona.