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A Review on Genus Paramacrobiotus

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17 July 2023

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Abstract
Paramacrobiotus species has been described from almost every corner of the world. To date 45 species have been reported from this genus. The species’ presence in different climatic conditions and habitat provides evidence of their adaptation to various harsh environments. In this review, we provide a concise summary of changes observed due to various cryptobiotic conditions in many species of this genus, geographical distribution of all the species, feeding behaviour, life history, microbiome community, Wolbachia endosymbiont identification, reproduction, phylogeny and general taxonomy of the species from genus Paramacrobiotus. Furthermore, we provide a new diagnostic key to the genus Paramacrobiotus based on the morphological and morphometric characters of adults and eggs.
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Subject: Biology and Life Sciences  -   Other

1. Introduction

Tardigrade, also called water bears, is a phylum consisting of ca. 1,500 species 1–4 that inhabit terrestrial and aquatic environments throughout the world5. They are mostly found in mosses, lichens, soil, leaf litter, sediments and on aquatic plants5–7. The phylum consists of two classes, i.e., Heterotardigrada and Eutardigrada5. Eutardigrada is further divided into two limnoterrestrial orders, i.e., Apochela and Parachela. Moreover, order Parachela consists of various superfamilies and families, one of them being Macrobiotidae (Thulin, 1928)8 with genus Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar and Wolf, 20099. The genus was erected in 2009 from the genus Macrobiotus and till date 45 species have been described: Paramacrobiotus alekseevi (Tumanov, 2005)10, Pam. arduus Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 201911, Pam. areolatus (Murray, 1907)12, Pam. beotiae (Durante Pasa & Maucci, 1979)13, Pam. celsus Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 201911, Pam. centesimus (Pilato, 2000)14, Pam. chieregoi (Maucci & Durante Pasa, 1980)15, Pam. corgatensis (Pilato, Binda & Lisi, 2004)16, Pam. csotiensis (Iharos, 1966)17, Pam. danielae (Pilato, Binda, Napolitano & Moncada, 2001)18, Pam. danielisae (Pilato, Binda & Lisi, 2006)19, Pam. depressus Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 201911, Pam. derkai (Degma, Michalczyk & Kaczmarek, 2008)20, Pam. experimentalis Kaczmarek, Mioduchowska, Poprawa & Roszkowska, 202021, Pam. fairbanksi Schill, Förster, Dandekar & Wolf, 201022, Pam. filipi Dudziak, Stec & Michalczyk 202023, Pam. gadabouti Kayastha, Stec, Mioduchowska and Kaczmarek 202324, Pam. garynahi (Kaczmarek, Michalczyk & Diduszko, 2005)25, Pam. gerlachae (Pilato, Binda & Lisi, 2004)16, Pam. halei (Bartels, Pilato, Lisi & Nelson, 2009)26, Pam. hapukuensis (Pilato, Binda & Lisi, 2006)19, Pam. huziori (Michalczyk & Kaczmarek, 2006)27, Pam. intii Kaczmarek, Cytan, Zawierucha, Diduszko & Michalczyk, 201428, Pam. kenianus Schill, Förster, Dandekar & Wolf, 201022, Pam. klymenki Pilato, Kiosya, Lisi & Sabella, 201229, Pam. lachowskae Stec, Roszkowska, Kaczmarek & Michalczyk, 201830, Pam. lorenae (Biserov, 1996)31, Pam. magdalenae (Michalczyk & Kaczmarek, 2006)27, Pam. metropolitanus Sugiura, Matsumoto & Kunieda, 202232 Pam. palaui Schill, Förster, Dandekar & Wolf, 201022, Pam. peteri (Pilato, Claxton & Binda, 1989)33, Pam. pius Lisi, Binda & Pilato, 201634, Pam. priviterae (Binda, Pilato, Moncada & Napolitano, 2001)35, Pam. richtersi (Murray, 1911)36, Pam. rioplatensis (Claps & Rossi, 1997)37, Pam. sagani Daza, Caicedo, Lisi & Quiroga, 201738, Pam. savai (Binda & Pilato, 2001)39, Pam. sklodowskae (Michalczyk, Kaczmarek & Węglarska, 2006)40, Pam. spatialis Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 201911, Pam. spinosus Kaczmarek, Gawlak, Bartels, Nelson & Roszkowska, 201741, Pam. submorulatus (Iharos, 1966)17, Pam. tonollii (Ramazzotti, 1956)42, Pam. vanescens (Pilato, Binda & Catanzaro, 1991)43, Pam. walteri (Biserov, 1997/98)44 and Pam. wauensis (Iharos, 1973)45. Furthermore, the genus is divided into two species groups, i.e., richtersi group with presence of microplacoid within the pharynx, and areolatus group without microplacoid within the pharynx. In turn, Kaczmarek et al.41 proposed to separate subgenera for which specific names were clarified by Marley et al.46. However, the two subgenera are not valid according to Guidetti et al.11 and Stec et al.47.
In this paper we summarise the data on taxonomy, distribution, mode of reproduction, microbiome study, feeding behaviour, life history, morphological taxonomy, phylogeny and cryptobiotic studies along with new key for species identification in genus Paramacrobiotus.

2. Cryptobiosis

A stage of an organism's life known as cryptobiosis is one in which no activity is apparent48. Many organisms go through cryptobiosis to survive the harsh environmental conditions they encounter 49–51. These conditions can include anhydrobiosis (lack of water), anoxybiosis (lack of oxygen), cryobiosis (low temperature), or osmobiosis (change in osmotic conditions). Tardigrades have a remarkable capacity for undergoing and surviving several types of cryptobiosis 48,52. The majority of anhydrobiosis, or absence of water, has been studied in the species of the genus Paramacrobiotus, although there has also been research on famine, freezing, and bet-hedging53–57. Reuner et al.53 studied how the influence of starvation and anhydrobiosis affects the size and number of storage cells in Paramacrobiotus tonollii to understand the energetic side of anhydrobiosis. Starving Pam. tonollii for seven days led to reduction in storage cell size by 46.41% but no significant reduction in storage cell number was observed. Furthermore, when storage cells size and number were investigated after inducing anhydrobiosis for seven days where no significant changes in storage cell size and number of Pam. tonollii was observed. Also, the mortality was checked using prolonged starvation and Pam. tonollii reached 50% mortality after 30 days. Likewise, Rizzo et al.54 investigated antioxidant defenses (capable of counteracting reactive oxygen species (ROS)) in Pam. richtersi in both active and dehydrated states. Activity of several antioxidant enzymes, the fatty acid composition and heat shock protein (Hsp) expression were compared in these two states. The increase in both antioxidant enzyme (superoxide dismutase due to induction of both glutathione peroxidase and glutathione during desiccation) and the fatty acid composition (polyunsaturated fatty acids and the amount of thiobarbituric acid reactive substances) were observed in desiccated Pam. richtersi specimens but no significant differences in the relative level of heat shock proteins were observed (Hsp70 and Hsp90). In addition, Tsujimoto et al.55 performed a study where the production of reactive oxygen species and involvement of bioprotectants during anhydrobiosis in Pam. spatialis was investigated. The study provides evidence of increase in ROS production relative to time spent in anhydrobiosis which is due to oxidative stress in the animals. Using RNA interference, involvement of bioprotectants, including those combating ROS was assessed. As Rizzo et al.54 concluded the role of glutathione peroxidase in desiccation in Pam. richtersi, this gene was targeted and what was observed is that glutathione peroxidase gene compromised survival during drying and rehydration of Pam. spatialis. This furthermore strengthened the evidence that glutathione reductase and catalase play important roles during desiccation and rehydration. Also, involvement of aquaporins 3 and 10 during rehydration of Pam. spatialis was observed. And recently Roszkowska et al.57 study the length that different tardigrades survive in the anhydrobiotic state including Pam. experimentalis. The study concludes that anhydrobiotic competence is dependent on habitat instead of nutritional behavior and the time taken to return to activity after anhydrobiosis is dependent upon the length of the anhydrobiosis.

3. Distribution

The distribution of species from this genus shows worldwide distribution. The distribution of all 45 species in genus Paramacrobiotus till date is presented in SM.01 and Figure 1.

4. Feeding behaviour

The tardigrade species Paramacrobiotus are omnivorous, consumes a variety of organisms, including certain cyanobacteria, algae, and fungi, as well as the rotifer Lecane, the nematode Caenorhabditis, and small juvenile tardigrades. Additionally, the diet of adults and juveniles eat is different: adults favour rotifers and nematodes, whereas juveniles favour unicellular green algae. Moreover, juveniles suck out all of them, including algal cells, animal food, and fungal cells, in contrast to adults who only consume entire fungal and algal cells58.

5. Life history

Life history refers to total life span, development, reproduction and death of an organism59. The life history list in case of tardigrades consists of age at first oviposition, clutch size, fecundity, hatching percentage, hatching success, lifespan, moulting number and total number of ovipositions60,61. The lifespan differs from species to species in case of tardigrades62. The life history of only a few Paramacrobiotus species have been reported till date. Namely, Pam. fairbanski with an average lifespan of 137.3 ± 136.4 days and 194.9 ± 164.4 days respectively and age at first oviposition of 70.7 ± 19.4 days and 76.9 ± 16.4 days respectively63; Pam. kenianus with average lifespan of 125 ± 35 days and 141 ± 54 days respectively, maximum life span of 204 days and 212 days respectively and age at first oviposition of 10 days and10 days respectively60; Pam. metropolitanus with juveniles hatching in 12–20 days, first oviposition in 11–13 days after hatching64; Pam. palaui with average lifespan of 97 ± 31 days, maximum life span of 187 days and age at first oviposition of 10 days60; Pam. richtersi with age at first oviposition of 64.2 ± 1.7 days65; Pam. tonollii with average lifespan of 69.0 ± 45.1 days and maximum life span of 237 days and age at first oviposition 24.4 ± 4.4 days62.

6. Microbiome

The microbiome represents the entire community of microorganisms, including fungi, protists, bacteria, archaea, as well as viruses, that inhabit all known metazoan species. The bacterial component of the microbiome community plays crucial roles in multiple aspects of ecdysozoan host life, such as behavior, metabolism, development, immunity, or pathogen defense, thereby regulating the functioning of the entire organism66,67. Conversely, it has also been demonstrated that the host's phylogeny68 and diet69 have significant impacts on the overall microbial composition. Indeed, many metazoan species appear to harbor their own specific microbiome community70. However, our understanding of the microbiome composition of Tardigrada, based on next-generation sequencing methods (NGS) targeting the standard 16S rRNA bacterial barcoding gene fragment, is limited to a very small number of published articles71–77.
In the case of species from the genus Paramacrobiotus, the microbiomes of a few species have been studied to date. In 2018, Vecchi et al.71 described the bacterial communities associated with six limno-terrestrial tardigrade taxa, one of which was Pam. areolatus. The study revealed that the microbial community was mainly composed of Proteobacteria and Bacteroidetes. Interestingly, certain classified Operational Taxonomic Units (OTUs) showed variations among species from geographically distant samples, indicating the presence of specific bacterial communities in each species. However, in all the investigated species' microbiome profiles, the order Rickettsiales was consistently identified. This order belongs to the class Alphaproteobacteria and is characterized by both pathogens and intracellular mutualists78. There were two distinct patterns in the diversity observed between tardigrades and their substrates, indicating significantly less microbial diversity in tardigrades compared to their substrates. This phenomenon may be attributed to tardigrades selectively associating with specific microbial communities that promote the growth of certain bacterial species while inhibiting others. Another hypothesis suggests that substrates, being complex matrices with wide surface areas and volumes, can support a high bacterial biomass, resulting in a vast and complex microbial community.
Similarly, Kaczmarek et al.21 conducted a microbiome analysis on two populations of Pam. experimentalis from Madagascar and their laboratory culture environment. These populations of Pam. experimentalis had been maintained in laboratory culture for two years. The most abundant phylum in all samples was Proteobacteria. Firmicutes was the second most dominant phylum in both Pam. experimentalis populations, while Bacteroides was the second most dominant phylum in the laboratory habitat. With the exception of the phyla Verrucomicrobia and Saccharibacteria, which were not found in the tardigrade microbiome, all identified taxa in the Pam. experimentalis microbiome community and laboratory culture environment were widespread and had comparable abundances. This confirms that the tardigrade microbiome significantly differs in composition from the bacteria inhabiting their environment. Moreover, within the microbiome of Pam. experimentalis, Operational Taxonomic Units (OTUs) classified as potential endosymbionts belonging to the order Rickettsiales were identified. The absence of Rickettsiales OTUs in the environment of the studied species further supports the close association of these bacteria with their host.
Furthermore, Mioduchowska et al.73 conducted a study to investigate whether tardigrade species are infected with bacterial endosymbionts belonging to the genus Wolbachia. The analysis included Pam. fairbanksi and Paramacrobiotus sp. In the study Proteobacteria, Firmicutes, and Actinobacteria as the three most prevalent phyla among the analyzed tardigrades, including species outside the genus Paramacrobiotus, were identified. However, the focus of the study was on potential tardigrade endosymbionts, particularly Operational Taxonomic Units (OTUs) from the order Rickettsiales and the genus Wolbachia. Both Rickettsiales and Wolbachia were detected in adult Paramacrobiotus sp., while only Rickettsiales were found in Pam. fairbanksi eggs. Adult Pam. fairbanksi did not have either Wolbachia or Rickettsiales infections. The genus Wolbachia is an intracellular bacterium belonging to the order Rickettsiales and it infects various invertebrates, particularly terrestrial insects79. However, recent studies have identified infections of this bacterial endosymbiont in various freshwater invertebrate species77,80,81. Generally, this bacterium is transmitted vertically from mother to offspring and/or through horizontal transfer directly from the environment or between different hosts82. Subsequently, Wolbachia manipulates host reproduction by inducing parthenogenesis, feminization, male killing, or cytoplasmic incompatibility83,84.
In 2023, Mioduchowska et al.77 described new molecular and bioinformatic tools for detecting Wolbachia in freshwater invertebrates. In this study, Wolbachia was detected in Pam. experimentalis, which were the same isolates analysed by Kaczmarek et al.72. Phylogenetic analysis of the obtained bacterial sequences allowed for their classification within the differentiated supergroup A of the genus Wolbachia. The discovery of Wolbachia in tardigrades opens new frontiers in understanding the Wolbachia-driven biology and ecology of Tardigrada.

7. Reproduction

Reproduction refers to the process where every organism known produces offspring either sexually or asexually. In case of tardigrades, they reproduce only through gametes via many different patterns i.e. dioecious (separate male and female), hermaphroditic (single animal with both male and female reproductive parts) or parthenogenetic (form of asexual reproduction)85. The genus Paramacrobiotus consists of both bisexual and unisexual species/populations. Pam. richtersi is both bisexual and unisexual from Italy; according to modern taxonomy they probably constitute a distinct species, Pam. areolatus population from Italy is bisexual and population from Svalbard is unisexual, Pam. tonolli from the USA is bisexual, Pam. fairbanksi is unisexual from various locations as Antarctic, Italy, Poland, Spain and USA, Pam. kenianus from Kenya is unisexual and Pam. palaui from Micronesia is unisexual, Pam. depressus from Italy is bisexual, Pam. celsus from Italy is bisexual, Pam. spatialis from Italy is bisexual, Pam. arduus from Italy is bisexual, Pam. experimentalis from Madagascar is bisexual and Pam. gadabouti is unisexual from various locations in Portugal, Australia, France and Tunisia. Out of 45, mode of reproduction for only 18 species are known (SM.01). Also, Guidetti et al.11 suggests the mode of reproduction being related to constrained or wide distribution of the species. The amphimictic species displays a very constrained or punctiform distribution, in contrast to the parthenogenetic species' extremely extensive spread and presence over multiple continents. The difference in the ability for dispersal linked to the two modes of reproduction can be used to explain why apomictic and amphimictic populations are distributed differently.

8. Morphological taxonomy

The genus Paramacrobiotus is divided into two morphologically distinct species groups: areolatus (species without a microplacoid or with rudimentary structures in the place of microplacoid in the pharynx) and richtersi (species with a microplacoid in the pharynx)( e.g.23,28). It was suggested that initially the microplacoid was present, however it was lost in some species from the areolatus group. But, the opposite situation, in which the microplacoid gradually appears, is also possible41. For example, in Pam vanescens the microplacoid suggests a gradual reduction. In turn, in Pam. areolatus and Pam. centesimus the microplacoid is generally absent, but a thin cuticular thickening is present in the place where microplacoid should be normally present and can be considered as rudimentary microplacoid14,47. Although, the presence or absence of microplacoid seems to be a clear morphological character dividing genus Paramacrobiotus into two separate phylogenetic lineages (which was suggested by Kaczmarek et al.41) the genetic studies did not confirm this11,47.
At present 45 species are formally attributed to the genus Paramacrobiotus and 13 belong to areolatus group and 32 richtersi group. They can be further divided into smaller groups based on egg types. In total, seven types of eggs were identified. However, two of them (areolatus and richtersi types) are the most common and occur in 37 species (ca. 82%). In the next two species huziori type of eggs are present (ca. 5%). The other types of eggs (i.e. beotiae, chieregoi, csotiensis, tonollii and submorulatus) were identified only in single taxa (for details of egg morphology see Kaczmarek et al.41). What is more, eggs are unknown for one species i.e. Pam. wauensis.
In recent years two very important for taxonomy of the entire genus, species Pam. areolatus and Pam. richtersi were integratively redescribed11,47. Another species Pam. fairbanksi described based, mostly, on genetic data was also morphometrically well characterized few years ago21. However, a few Paramacrobiotus species still need a redescription based on type material or on additional material from type localities. Descriptions of Pam. beotiae, Pam. chieregoi, Pam. csotiensis, Pam. rioplatensis, Pam. submorulatus, Pam. tonollii and Pam. wauensis are inaccurate and some important morphological information are lacking.
Another two species, i.e., Pam. kenianus and Pam. palaui are cryptic taxa described mostly based on genetic data without morphological differential diagnosis22.
Descriptions of the other Paramacrobiotus species more or less complete, but in most of them exact morphometric data of claws, buccal tubes placoids and above all genetic data are lacking (see Table 1 and SM.01). Based on all the abovementioned doubts, 3 species, i.e., Pam. kenianus, Pam. palaui and Pam. wauensis were not included to the key.

9. Molecular taxonomy

Molecular markers serve as valuable tools for species identification. In the integrative taxonomy of Tardigrada, four DNA fragments with different mutation rates are commonly used: two conservative nuclear ribosomal subunit genes, namely 18S rRNA (the small ribosome subunit) and 28S rRNA (the large ribosome subunit), the noncoding nuclear ITS2 fragment (the internal transcribed spacer-2) with high evolution rates, and the protein coding mitochondrial COI barcode gene (the cytochrome oxidase subunit I) with an intermediate effective mutation rate (e.g., Kaczmarek et al.72). The COI mtDNA molecular marker, in particular, has been recommended for DNA barcoding purposes (http://www.barcodinglife.org), such as rapid species identification, discrimination between cryptic species, and resolving phylogenetic relationships among closely related species86,87. To gain additional insights into the phylogenetic relationships within the genus Paramacrobiotus, an analysis based on COI mtDNA was conducted. This analysis was performed to supplement the information obtained from previous studies using four molecular markers24.
Due to ongoing revisions and redescriptions of Paramacrobiotus species, studies are becoming more accessible, leading us to anticipate that the species diversity within the genus is greatly underestimated11,23. One significant challenge that needs to be addressed in future studies is the lack of available barcodes. Despite the designation of 45 species to the genus Paramacrobiotus, not all species have available barcode sequences. In this study, we aimed to estimate the phylogenetic relationships among all Paramacrobiotus species (including taxa designated as "cf." – meaning "compare with" and "aff." – meaning "similar to") for which COI barcode sequences are available in the GenBank database. We used the COI sequence of Milnesium berladnicorum Ciobanu, Zawierucha, Moglan & Kaczmarek, 201488 as outgroups to construct the most reliable evolutionary tree. To determine the most appropriate model of sequence evolution, we applied jModelTest v. 2.1.489 with both the Bayesian Information Criterion (BIC) and the Akaike Information Criterion (AIC)90. The GTR + G (Time Reversible model with gamma distributed rate heterogeneity) was selected as the best-fit evolutionary model. The phylogenetic tree was constructed using Bayesian inference (BI) analysis with the program MrBayes 391, following the settings described by Mioduchowska et al.92. The alignment of COI barcode sequences resulted in 574 characters, with 270 variable sites and 241 parsimony informative sites. Uncorrected pairwise distances (p-distances) were calculated using MEGA X93.
The binary model of phylogenetic relationships, which involves reconstructing gene trees from sequence data, allows us to gain insights into the speciation history of species94. However, in our analysis of barcode sequences, we observed speciation events that resulted in polytomies within the phylogeny of the genus Paramacrobiotus (Figure 2). This means that more than two descendants were observed from certain nodes95. The presence of unresolved nodes in a polytomic multifurcating tree indicates a lack of signal in the data to resolve relationships within the genus Paramacrobiotus. This observation is partially consistent with previous studies, where both groups of richtersi and areolatus were described as polyphyletic11,47. However, in the work by Kayastha et al.24, the interrelationships of the genus Paramacrobiotus were not depicted as a polytomy when two conservative coding nuclear molecular markers (18S rRNA and 28S rRNA) and a noncoding nuclear marker with high evolution rates (ITS2) were included in the analysis. As a result, the phylogenetic relationships within the genus Paramacrobiotus were resolved. Interestingly, other examples of polytomies in Tardigrada gene trees based on nuclear molecular markers have also been observed96.
The genetic p-distances between the analyzed COI barcode sequences of Paramacrobiotus species ranged from 16% to 27%, indicating different species (Table 2). However, it was shown that there are very low genetic differences, i.e., a p-distance of 0.3%, between Pam. aff. richtersii from Tunisia (GenBank: MH676016) and Pam. gadabouti from Portugal (GenBank: OP394113), suggesting they belong to the same species (Table 2). This finding is consistent with the work by Kayastha et al. 24, where both species were described as Pam. gadabouti. No genetic differences were found between Pam. aff. richtersi from Madagascar (GenBank: MH676008) and Pam. experimentalis from Madagascar (GenBank: MN097836) (Table 2). Both sequences represented Pam. experimentalis, which is also consistent with the previous study (Kayastha et al.24). Moreover, we found very low genetic differences, i.e., a p-distance of 2.1%, between Pam. arduus from Italy (GenBank: MK041020) and Pam. aff. arduus from Italy (GenBank: MK041022), indicating the same species (Table 2).
The text continues here (Figure 2 and Table 2).

10. Key for species identification

1. Microplacoid present (richtersi group) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
–. Microplacoid absent (areolatus group) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2. Cuticular pattern on dorsal side of the body present and visible in LM (PCM and/or DIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
–. Cuticle on dorsal side of the body smooth or cuticular pattern not visible in LM (PCM and/or DIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Eggs of areolatus type. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. danielae
–. Eggs of richtersi type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 4
4. Eyes present, lunules under claws IV dentate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. corgatensis
–. Eyes absent, lunules under claws IV smooth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5. Dorsal cuticle covered with very small circular or elongated tubercles, egg processes less than 14.5 μm height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. halei
–. Dorsal cuticle covered with small dots (granules) or small polygons, egg processes more than 15.5 μm height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6. Dorsal cuticle covered with small dots (granules) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. vanescens
–. Dorsal cuticle covered with small polygons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. danielisae
7. Areolation between egg processes absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
–. Areolation between egg processes present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
8. Lunules under claws IV dentate, eggs of beotiae type . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . Pam. beotiae
–. Lunules under claws IV smooth, egg of chieregoi type . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. chieregoi
9. Eggs of submorulatus type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . Pam. submorulatus
–. Eggs of richtersi or areolatus type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
10. Eggs of richtersi type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
–. Eggs of areolatus type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
11. Only five or six areoles present around each egg process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
–. The number of areoles around each egg process larger than six . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 18
12. Eyes present. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. priviterae
–. Eyes absent. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
13. Granulation on leg I-III present. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .14
–. Granulation on legs I-III absent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. depressus
14. The pt values of the macroplacoid length less than 43.5. . .. . . .. . . . . . . . . . . . . . . . . . . . . . . Pam. pius
–. The pt values of the macroplacoid length more than 49.0. . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 15
15. Egg process jagged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . 16
–. Egg process not jagged . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
16. Egg processes height less than 15.0 μm and parthenogenetic mode of reproduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. fairbanksi
–. Egg processes height more than 15.1 μm and bisexual mode of reproduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. celsus
17. Egg diameter without processes less than 62.5 μm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. arduus
–. Egg diameter without processes more than 65.0 μm. . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. spatialis
18. Eyes present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
–. Eyes absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
19. Granulation on legs I-III present . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
–. Granulation on legs I-III absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. magdalenae
20. Egg bare diameter less than 87.9 μm, egg process height more than 15 μm, egg processes hemispherical with blunt terminal part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. sklodowskae
–. Egg bare diameter more than 92.0 μm, egg process height less than 13.5 μm, egg processes hemispherical with cylindrical indented apices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. sagani
21. Lunules under claws IV dentate. . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .Pam. alekseevi
–. Lunules under claws IV smooth. . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
22. Egg processes with cap-like vesicular structures on the top . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
– Egg processes without cap-like vesicular structures on the top. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
23. Egg processes with elongated terminal portion, second macroplacoid length less than 6.5 μm, pt values of second macroplacoid length less than 14.0, pt values of macroplacoid row length less than 59.0, placoid row length less than 34.5 μm and pt values of placoid row length less than 74.0. . .. ... . . . . . . . . . . . . . . . .. ... . . . . . ... .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. filipi
– Egg processes without elongated terminal portion, second macroplacoid length 7.0 μm or more, pt values of second macroplacoid length more than 15.0, pt values of macroplacoid row length more than 60.0, placoid row length more than 34.9 μm and pt values of placoid row length more than 77.5. . . . . .. . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. gadabouti
24. Egg processes with long, thin and flexible terminal portion and egg process height more than 24.5 μm . . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. lorenae
– Egg processes without long, thin and flexible terminal portions and egg process height less than 22.5 μm . . . . . . . .. . . . . . .. . . . .. . . . . . .. . . . . . .. . . . .. . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . Pam. richtersi
25. Cuticle with oval pores, egg processes with cap-like structure on the top and clearly narrower under caps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. garynahi
–. Cuticle without oval pores, egg processes without cap-like structure on the top and without narrowing at the top . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
26. Egg processes hemispherical with blunt apex not divided and without elongated terminal part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. savai
–. Egg processes different . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 27
27. Egg processes with long flexible spines on the top . . . . . . . . . . . . . . . .. . . . . . . . .Pam. rioplatensis
–. Egg processes without long flexible spines on the top . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
28. Egg processes base width less than 12.5 μm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. peteri
–. Egg processes base width more than 13.0 μm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
29. Granulation on IVth pair of legs absent and egg processes height more than 15.5 μm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. hapukuensis
–. Granulation on IVth pair of legs present and egg processes height less than 15.0 μm . 30
30. Presence of wrinkled surface on the egg areolae and the absence of cuticular bulge on inner surface of claws I–III. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Pam. experimentalis
–. Lack of wrinkled surface on the egg areolae and the presence of cuticular bulge on inner surface of claws I–III. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . .. . .. . . . . . . . . . Pam. metropolitanus
31. Egg of csotiensis type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. csotiensis
–. Eggs of areolatus, huziori, tonollii or richtersi type . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 32
32. Eggs of tonollii type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. tonollii
–. Eggs of areolatus, huziori or richtersi type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .33
33. The egg areolation of the huziori type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
–. Eggs of richtersi or areolatus type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
34. Only one row of larger teeth present in the second band in the oral cavity, the distances between all macroplacoids are approximately the same, accessory points well developed but not protruding high above the primary branch, diameter of bases of egg processes approximately equal to or slightly smaller than their height, 9–11 processes on egg circumference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . Pam. huziori
–. A row of larger teeth and a posterior band of small granules/conical teeth present in the second band of teeth in the oral cavity, the second macroplacoid situated closer to the first than to the third macroplacoid, accessory points extremely well developed, protruding high above the primary branch, diameter of bases of egg processes greater than their height, 12–16 processes on egg circumference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. derkai
35. Eggs of richtersi type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. spinosus
–. Eggs of areolatus type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
36. The first/anterior band of teeth visible under PCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
–. The first/anterior band of teeth absent or not visible under PCM . . . . . . . . . . . . . . . . . . . . .Pam. intii
37. Lunules under claws IV smooth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
–. Lunules under claws IV dentate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
38. Eyes present, macroplacoid length sequence 2<3<1, full egg diameter more than 93.0 μm and egg process height more than 17.5 μm. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pam. lachowskae
–. Eyes absent, macroplacoid length sequence 2<1<3, full egg diameter less than 92.0 μm and egg process height less than 11.5 μm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Pam. centesimus
39. Eyes present, macroplacoid length sequence 2<1<3 and egg processes elongated . . . . . . . . . . . . . .40
–. Eyes absent, macroplacoid length sequence 2<3<1 and egg processes short. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. klymenki
40. Egg process height more than 26.5 μm and egg process surface smooth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pam. areolatus
–. Egg process height less than 17.5 μm and egg process surface apically covered by irregular granulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . Pam. walteri

5. Conclusions

The genus Paramacrobiotus shows cosmopolitan distribution with presence of both bisexual and parthenogenetic species. Although the integrative descriptions and redescriptions are improving the overall situation and allowing for fresh opportunities for detailed study, the phylogeny of the genus Paramacrobiotus seems to be unresolved. Also, there are many other studies regarding life-history, cryptobiotic abilities and microbiome community as well as bacterial endosymbiont infections identification, which are lacking, and such studies are required for the advancement of tardigrade knowledge in general.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org. SM.01 Locations, mode of reproduction and presence of genetic data for all the Paramacrobiotus species.

Author Contributions

Conceptualization, P.K. and Ł.K.; methodology, P.K. and M.M.; formal analysis, P.K. and M.M.; investigation, P.K.; data curation, P.K.; writing—original draft preparation, P.K.; writing—review and editing, P.K., M.M. and Ł.K; visualization, P.K. and M.M.; supervision, Ł.K. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

All the DNA sequences data are from GenBank.

Acknowledgments

Studies have been partially conducted in the framework of activities of BARg (Biodiversity and Astrobiology Research group). PK is scholarship holder of Passport to the future - Interdisci-plinary doctoral studies at the Faculty of Biology, Adam Mickiewicz University, Poznań POWR.03.02.00-00-I006/17. The work of MM was supported by National Science Centre, Poland, grant no. 2021/43/D/NZ8/00344 and grant no. 1220/146/2021 from the Small Grants Pro-gramme of the University of Gdansk (i.e., Ugrants-first competition). Tomasz Bartylak for helping with QGIS.

Conflicts of Interest

The authors declare no competing interests.

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Figure 1. Distribution of all the species in genus Paramacrobiotus (co-ordinates present in SM.01). (Map prepared using QGIS ver. 3.28.0-Firenze).
Figure 1. Distribution of all the species in genus Paramacrobiotus (co-ordinates present in SM.01). (Map prepared using QGIS ver. 3.28.0-Firenze).
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Figure 2. Phylogenetic relationships of the genus Paramacrobiotus constructed based on the COI barcode sequences obtained from the GenBank database. The GenBank accession numbers are given in parentheses. In turn, locations of identified species given in abbreviations: JP – Japan; PL – Poland; HU – Hungary; IT – Italy; MG – Madagascar; MY – Malaysia; BR – Brazil; PT – Portugal; TN – Tunisia; NO – Norway; IE – Ireland; CO – Colombia; US – United States. The numbers at the branches represent Bayesian posterior probabilities. The COI sequence of Milnesium berladnicorum was used as an outgroup.
Figure 2. Phylogenetic relationships of the genus Paramacrobiotus constructed based on the COI barcode sequences obtained from the GenBank database. The GenBank accession numbers are given in parentheses. In turn, locations of identified species given in abbreviations: JP – Japan; PL – Poland; HU – Hungary; IT – Italy; MG – Madagascar; MY – Malaysia; BR – Brazil; PT – Portugal; TN – Tunisia; NO – Norway; IE – Ireland; CO – Colombia; US – United States. The numbers at the branches represent Bayesian posterior probabilities. The COI sequence of Milnesium berladnicorum was used as an outgroup.
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Table 1. Selected morphological characters of the known species of genus Paramacrobiotus.
Table 1. Selected morphological characters of the known species of genus Paramacrobiotus.
Species Cuticle Oral Cavity Armature Eyes Lunules IV Granulation on Legs Type of Egg Egg process height (min. and max. values in μm) Egg process base width (min. and max. values in μm) Egg process shape Number of processes on circumference
Paramacrobiotus alekseevi smooth I–III absent dentate IV richtersi 11.8–21.8 13.3–22.9 with cap 10–12
Paramacrobiotus arduus smooth I–III absent smooth I–IV richtersi 12.1–18.3 10.4–16.3 conical 16–21
Paramacrobiotus areolatus smooth I–III present crenate I–IV areolatus 20.0–28.0 19.0–22.0 conical ?
Paramacrobiotus beotiae smooth I–III absent dentate ? beotiae up to 16.0 ? spines ?
Paramacrobiotus celsus smooth I–III absent smooth I–IV richtersi 15.2–19.1 14.3–18.2 conical (jagged) 15–19
Paramacrobiotus centesimus smooth I–III absent smooth I–IV areolatus 7.0–11.0 ? conical 11–12
Paramacrobiotus chieregoi smooth I–III absent smooth ? chieregoi ? ? elongated 14
Paramacrobiotus corgatensis sculptured I–III present dentate ? richtersi 20.0–25.0 18.0–24.0 conical (jagged) 8–11
Paramacrobiotus csotiensis smooth II–III present ? ? csotiensis ? ? blunt ?
Paramacrobiotus danielae sculptured I–III present smooth ? areolatus 14.5 24.7 conical ?
Paramacrobiotus danielisae sculptured I–III absent smooth ? richtersi 17.3–23.0 17.5–20.0 conical 9–10
Paramacrobiotus depressus smooth I–III absent smooth IV richtersi 9.3–12.4 12.4–15.2 conical 16–23
Paramacrobiotus derkai smooth I–III present smooth I–IV huziori 8.0–17.1 12.5–28.3 conical 12–16
Paramacrobiotus experimentalis smooth I–III absent smooth IV areolatus 10.3 – 14.9 13.8 – 19.4 conical 10–12
Paramacrobiotus fairbanksi smooth I–III absent smooth I–IV richtersi 10.9 – 14.9 10.9 – 20.8 conical (jagged) ?
Paramacrobiotus filipi granulation I–III absent smooth I–IV richtersi 17.8–25.2 11.7–21.7 elongated with disc 10–11
Paramacrobiotus gadabouti smooth I–III absent smooth IV richtersi 12.1–23.7 15.0–25.5 truncated cones 11–13
Paramacrobiotus garynahi with pores I–III absent smooth I-IV areolatus 18.0–30.0 20.0–42.0 with cap 10–13
Paramacrobiotus gerlachae smooth I–III absent smooth IV richtersi 11.8–14.5 16.8–18.7 blunt ?
Paramacrobiotus halei sculptured I–III absent ? I-IV richtersi 11.0–14.0 22.0–23.5 blunt 11
Paramacrobiotus hapukuensis smooth I–III absent smooth absent areolatus 15.7–21.1 14.8–16.6 elongated 10
Paramacrobiotus huziori smooth I–III present smooth I–IV huziori 20.0–33.0 20.0–30.0 conical 9–11
Paramacrobiotus intii smooth II–III present dentate I–IV areolatus 15.4–24.4 22.0–34.0 conical 9–10
Paramacrobiotus kenianus smooth ? present ? ? richtersi 13.5 ± 1.9 19.7 ± 2.7 conical 17.7 ± 3.6
Paramacrobiotus klymenki smooth I–III absent dentate I–IV areolatus 14.5–18.5 16.4–18.2 conical 10–11
Paramacrobiotus lachowskae smooth I–III present smooth I–IV areolatus 17.6–32.1 8.1–17.7 dome with filaments 8–14
Paramacrobiotus lorenae smooth I–III absent smooth I-IV richtersi 25.0–42.2 17.8–23.0 elongated ?
Paramacrobiotus magdalenae smooth I–III present smooth IV richtersi 13.0–25.0 16.2–21.0 conical 10–12
Paramacrobiotus metropolitanus smooth I–III absent smooth IV areolatus 7.4–14.6 9.8–21.1 conical 10–15
Paramacrobiotus palaui smooth ? present ? ? richtersi 10.2 ± 1.3 13.4 ± 1.3 conical 15.4 ± 1.4
Paramacrobiotus peteri smooth I–III absent smooth ? areolatus 10.0–14.0 9.0–12.0 conical (jagged) ?
Paramacrobiotus pius smooth I–III absent smooth I-IV richtersi up to 12.3 19.5–24.7 conical (jagged) 10
Paramacrobiotus priviterae smooth I–III present smooth I-IV richtersi 11.8–15.0 12.9–16.3 conical (jagged) ?
Paramacrobiotus richtersi smooth I–III absent smooth I-IV richtersi 17.1–22.1 17.2–22.2 conical 13–17
Paramacrobiotus rioplatensis smooth I–III present smooth ? areolatus ca. 4.6 ? elongated 17-19
Paramacrobiotus sagani granulation I–III present smooth I-IV richtersi 9.4–13.2 14.6–22.4 cylindrical, indented apices 10–13
Paramacrobiotus savai smooth I–III present smooth IV areolatus 12.0–18.0 16.7–18.5 blunt ?
Paramacrobiotus sklodowskae smooth I–III present smooth I-IV richtersi 16.0–17.5 20.5–23.5 blunt 10
Paramacrobiotus spatialis smooth I–III absent smooth I-IV richtersi 13-16 15.2–20.4 conical 15–23
Paramacrobiotus spinosus smooth I–III absent smooth I–IV richtersi 22.1–42.2 17.3–26.0 elongated (jagged) 10–11
Paramacrobiotus submorulatus smooth II–III present ? ? submorulatus 7.0–8.3 17.5–20.4 blunt 13
Paramacrobiotus tonollii smooth ? present smooth ? tonollii 32.0–35.0 ? conical 8–10
Paramacrobiotus vanescens smooth I–III absent ? I-IV richtersi 16.0–17.0 24.0–25.0 blunt (jagged) 9–12
Paramacrobiotus walteri smooth I–III present dentate I–IV areolatus 10.0–17.0 9.0–20.0 conical (jagged) ?
Paramacrobiotus wauensis smooth I– III absent ? ? ? ? ? ? ?
Table 2. Estimates of evolutionary divergence between COI barcode sequences based on p-distances.
Table 2. Estimates of evolutionary divergence between COI barcode sequences based on p-distances.
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