Unsupervised machine learning and geometric morphometrics as tools for the identification of inter and intraspecific variations in the Anopheles Maculipennis complex.

Geometric morphometric analysis was combined with two different unsupervised machine learning algorithms, UMAP and HDBSCAN, to visualize morphological differences in wing shape among and within four Anopheles sibling species (An. atroparvus, An. melanoon, An. maculipennis s.s. and An. daciae sp. inq.) of the Maculipennis complex in Northern Italy. Specifically, we evaluated: (1) wing shape variation among and within species; (2) the consistencies between groups of An. maculipennis s.s. and An. daciae sp. inq. identified based on COI sequences and wing shape variability; and (3) the spatial and temporal distribution of different morphotypes. UMAP detected at least 13 main patterns of variation in wing shape among the four analyzed species and mapped intraspecific morphological variations. The relationship between the most abundant COI haplotypes of An. daciae sp. inq. and shape ordination/variation was not significant. However, morphological variation within haplotypes was reported. HDBSCAN also recognized different clusters of morphotypes within An. daciae sp. inq. (12) and An. maculipennis s.s. (4). All morphotypes shared a similar pattern of variation in the subcostal vein, in the anal vein and in the radio-medial cross-vein of the wing. On the contrary, the marginal part of the wings remained unchanged in all clusters of both species. Any spatial-temporal significant difference was observed in the frequency of the identified morphotypes.  Our study demonstrated that machine learning algorithms are a useful tool combined with geometric morphometrics and suggest to deepen the analysis of inter and intra specific shape variability to evaluate evolutionary constrains related to wing functionality.

The puzzling mitochondrial phylogeography of the black soldier fly (Hermetia illucens), the commercially most important insect protein species.

BACKGROUND: The black soldier fly (Diptera: Stratiomyidae, Hermetia illucens) is renowned for its bioconversion ability of organic matter, and is the worldwide most widely used source of insect protein. Despite varying extensively in morphology, it is widely assumed that all black soldier flies belong to the same species, Hermetia illucens. We here screened about 600 field-collected and cultured flies from 39 countries and six biogeographic regions to test this assumption based on data for three genes (mitochondrial COI, nuclear ITS2 & 28S rDNA) and in order to gain insights into the phylogeography of the species. RESULTS: Our study reveals a surprisingly high level of intraspecific genetic diversity for the mitochondrial barcoding gene COI (divergences up to 4.9%). This level of variability is often associated with the presence of multiple species, but tested nuclear markers (ITS2 and 28S rDNA) were invariant and fly strain hybridization experiments under laboratory conditions revealed reproductive compatibility. COI haplotype diversity is not only very high in all biogeographic regions (56 distinct haplotypes in total), but also in breeding facilities and research centers from six continents (10 haplotypes: divergences up to 4.3%). The high genetic diversity in fly-breeding facilities is mostly likely due to many independent acquisitions of cultures via sharing and/or establishing new colonies from field-collected flies. However, explaining some of the observed diversity in several biogeographic regions is difficult given that the origin of the species is considered to be New World (32 distinct haplotypes) and one would expect severely reduced genetic diversity in the putatively non-native populations in the remaining biogeographic regions. However, distinct, private haplotypes are known from the Australasian (N = 1), Oriental (N = 4), and the Eastern Palearctic (N = 4) populations. We reviewed museum specimen records and conclude that the evidence for introductions is strong for the Western Palearctic and Afrotropical regions which lack distinct, private haplotypes. CONCLUSIONS: Based on the results of this paper, we urge the black soldier fly community to apply molecular characterization (genotyping) of the fly strains used in artificial fly-breeding and share these data in research publications as well as when sharing cultures. In addition, fast-evolving nuclear markers should be used to reconstruct the recent invasion history of the species.

Evaluation of the molecular variability and characteristics of Paramecium polycaryum and Paramecium nephridiatum, within subgenus Cypriostomum (Ciliophora, Protista).

Although some Paramecium species are suitable research objects in many areas of life sciences, the biodiversity structure of other species is almost unknown. In the current survey, we present a molecular analysis of 60 Cypriostomum strains, which for the first time allows for the study of intra- and interspecific relationships within that subgenus, as well as the assessment of the biogeography patterns of its morphospecies. Analysis of COI mtDNA variation revealed three main clades (separated from each other by approximately 130 nucleotide substitutions), each one with internal sub-clusters (differing by 30 to 70 substitutions - a similar range found between P. aurelia cryptic species and P. bursaria syngens). The first clade is represented exclusively by P. polycaryum; the second one includes only four strains identified as P. calkinsi. The third cluster seems to be paraphyletic, as it includes P. nephridiatum, P. woodruffi, and Eucandidatus P. hungarianum. Some strains, previously identified as P. calkinsi, had COI sequences identical or very similar to P. nephridiatum ones. Morphological reinvestigation of several such strains revealed common morphological features with P. nephridiatum. The paper contains new information concerning speciation within particular species, i.e. existence of cryptic species within P. polycaryum (three) and in P. nephridiatum (six).

Morphometric characteristics and COI haplotype diversity of Arctodiaptomus spinosus (Copedoda) populations in soda pans in Hungary.

Arctodiaptomus spinosus (Daday, 1891) is a characteristic species of the soda pan zooplankton in the Great Hungarian Plain. The biogeographical distribution of the species is interesting, since its range expands from the Pannonian Biogeographic region to the other side of the Carpathians, occurring in saline lakes in Eastern Anatolia, Armenia, Iran and in temporary waters in Ukraine. Our investigations focused on the morphometric characteristics and the COI haplotype diversity of four Hungarian populations in the Kiskunság area. We detected substantial morphological differences between the Böddi-szék population and the rest of the sampling sites, however considerable differences were not observable in the COI haplotypes in the populations. The 20 animals investigated for COI haplotypes belonged to the same haplotype network. Tajima's D indicated departures from the neutral Wright - Fisher population model and suggested population expansion. The genetic composition of Arctodiaptomus spinosus populations in the Kiskunság area is rather uniform.

Characteristics of parthenogenesis in Cacopsylla ledi (Flor, 1861) (Hemiptera, Sternorryncha, Psylloidea): cytological and molecular approaches.

Characteristics of parthenogenesis in Cacopsylla ledi (Flor, 1861) were analyzed using cytological and molecular approaches. In all three populations studied from Finland, i.e. Turku, Kustavi and Siikajoki, males were present at a low frequency but were absent from a population from Vorkuta, Russia. In a follow-up study conducted in the Turku population during 2010-2016, the initial frequency of males was ca. 10 % and showed no intraseasonal variation, but then dramatically decreased down to approximately 1-2 % level in seasons 2015-2016. Male meiosis was chiasmate with some traces of chromosomal fragmentation and subsequent fusions. In most females, metaphase in mature eggs included 39 univalent chromosomes which indicated apomictic triploidy. Only a small fraction of females was diploid with 13 chiasmate bivalents. The frequency of diploid females approximately equaled that of males. COI barcode analyses showed that triploid females (N = 57) and diploids (7 females and 5 males) displayed different haplotypes, demonstrating that triploid females reproduced via obligate parthenogenesis. The rarity of diploids, along with the lack of males' preference towards diploid females, suggested that most likely diploids were produced by rare triploid females which shared the same haplotype with the diploids (not found in the present analysis). Minimum haplotype diversity was detected in the Turku population, but it was much higher in Vorkuta with some indication for the mixed origin of the population. We suggest that functional diploids produced in a parthenogenetic population can give rise either to a new parthenogenetic lineage or even to a new bisexual species.