A Review on the Genus Paramacrobiotus (Tardigrada) with a New Diagnostic Key

Abstract

Paramacrobiotus species have been described in almost every corner of the world. To date, 45 species have been reported from this genus. Among which, 13 belong to the areolatus group (without a microplacoid) and 32 belong to the richtersi group (with a microplacoid). The species’ presence in different climatic conditions and habitats provides evidence of their adaptation to various harsh environments. The species of the genus are both bisexual (diploid) and parthenogenetic (triploid). The bisexual species have external fertilization. And they are omnivorous whose diet consists of certain cyanobacteria, algae, fungi, rotifers, nematodes and juvenile tardigrades. The life history of species from this genus varies from species to species. Because the species has a strong predilection for cryptobiosis, numerous investigations involving anhydrobiosis have been conducted utilizing specimens from varied Paramacrobiotus species to date. In this review, we provide a concise summary of changes observed due to various cryptobiotic conditions in many species of this genus, the geographical distribution of all the species, feeding behaviour, life history, microbiome community, Wolbachia endosymbiont identification, reproduction, phylogeny and general taxonomy of the species from the genus Paramacrobiotus. Furthermore, we provide a new diagnostic key to the genus Paramacrobiotus based on the morphological and morphometric characters of adults and eggs.

1. Introduction

Tardigrades, also called water bears, is a phylum consisting of ca. 1500 species [1,2,3,4] that inhabit terrestrial and aquatic environments throughout the world [5]. They are mostly found in mosses, lichens, soil, leaf litter, sediments and on aquatic plants [5,6,7]. The phylum consists of two classes, i.e., Heterotardigrada and Eutardigrada [5]. Eutardigrada is further divided into two limnoterrestrial orders, i.e., Apochela and Parachela. Moreover, the order Parachela consists of various superfamilies and families, one of them being Macrobiotidae (Thulin, 1928) [8] with the genus Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar and Wolf, 2009 [9]. The genus was erected in 2009 from the genus Macrobiotus. These two genera are distinguished by morphological characteristics such as egg processes’ shape (large and reticulated cones or trunk-cones in the genus Paramacrobiotus, smooth and inverted goblet shaped in the genus Macrobiotus). Next, only the genus Paramacrobiotus’ buccal armature has a posterior line of strong triangular or bicuspidal teeth. Furthermore always three, well-separated macroplacoids in the Paramacrobiotus species are present but mostly two, and in rare cases three with the first two very close, in Macrobiotus species are present. Also, cuticular pores are absent in Paramacrobiotus but present in Macrobiotus. Lastly, the shape of the spermatozoa in the Paramacrobiotus species is such that the head is thin and very long, up to 100 µm, and it is longer than the tail; in the Macrobiotus species, the head is strongly coiled and long but shorter than the tail, and it has a huge midpiece [9]. To date, 45 species have been described: Paramacrobiotus alekseevi (Tumanov, 2005) [10]; Pam. arduus Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 2019 [11]; Pam. areolatus (Murray, 1907) [12]; Pam. beotiae (Durante Pasa & Maucci, 1979) [13]; Pam. celsus Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 2019 [11]; 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, 2019 [11]; Pam. derkai (Degma, Michalczyk & Kaczmarek, 2008) [20]; Pam. experimentalis Kaczmarek, Mioduchowska, Poprawa & Roszkowska, 2020 [21]; Pam. fairbanksi Schill, Förster, Dandekar & Wolf, 2010 [22]; Pam. filipi Dudziak, Stec & Michalczyk 2020 [23]; Pam. gadabouti Kayastha, Stec, Mioduchowska and Kaczmarek 2023 [24]; 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, 2014 [28]; Pam. kenianus Schill, Förster, Dandekar & Wolf, 2010 [22]; Pam. klymenki Pilato, Kiosya, Lisi & Sabella, 2012 [29]; Pam. lachowskae Stec, Roszkowska, Kaczmarek & Michalczyk, 2018 [30]; Pam. lorenae (Biserov, 1996) [31]; Pam. magdalenae (Michalczyk & Kaczmarek, 2006) [27]; Pam. metropolitanus Sugiura, Matsumoto & Kunieda, 2022 [32] Pam. palaui Schill, Förster, Dandekar & Wolf, 2010 [22]; Pam. peteri (Pilato, Claxton & Binda, 1989) [33]; Pam. pius Lisi, Binda & Pilato, 2016 [34]; 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, 2017 [38]; Pam. savai (Binda & Pilato, 2001) [39]; Pam. sklodowskae (Michalczyk, Kaczmarek & Węglarska, 2006) [40]; Pam. spatialis Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 2019 [11]; Pam. spinosus Kaczmarek, Gawlak, Bartels, Nelson & Roszkowska, 2017 [41]; 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., the richtersi group, with the presence of a microplacoid within the pharynx, and the areolatus group, without a microplacoid within the pharynx. In turn, Kaczmarek et al. [41] proposed separating 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 summarize the data on the taxonomy, distribution, mode of reproduction, microbiome study, feeding behaviour, life history, morphological taxonomy, phylogeny and cryptobiotic studies, along with providing a new key for species identification in the genus Paramacrobiotus.

2. 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 the microplacoid was initially present but was lost in some species from the areolatus group. But, the opposite situation, in which the microplacoid gradually appeared, is also possible [41]. 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 the microplacoid should normally be present and can be considered as a rudimentary microplacoid [14,47]. Although the presence or absence of the microplacoid seems to be a clear morphological character dividing the genus Paramacrobiotus into two separate phylogenetic lineages (which was suggested by Kaczmarek et al. [41]), but genetic studies did not confirm this [11,47].

At present, 45 species are formally attributed to the genus Paramacrobiotus, 13 belong to the areolatus group, and 32 belong to the 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, the 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]). Furthermore, eggs are unknown for one species, Pam. wauensis.

In recent years, two very important species for taxonomy of the entire genus, Pam. areolatus and Pam. richtersi, were integratively redescribed [11,47]. Another species, Pam. fairbanksi, described based mostly on genetic data, was also morphometrically well characterized a few years ago [21]. However, a few Paramacrobiotus species still need a redescription based on the 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 informations are lacking.

Another two species, i.e., Pam. kenianus and Pam. palaui, are cryptic taxa described mostly based on genetic data without morphological differential diagnosis [22].

Descriptions of the other Paramacrobiotus species are more or less complete, but in most of them, exact morphometric data of claws and buccal tubes placoids and, above all, genetic data are lacking (see Table 1 and Supplementary Materials SM.01). Based on all the abovementioned doubts, three species, i.e., Pam. kenianus, Pam. palaui and Pam. wauensis, are not included in the key.

Table 1. Selected morphological characters of the known species of genus Paramacrobiotus (schematic illustrations of different types of egg process shapes presented in Figure 1).

Species	Cuticle	Number of Rows in Oral Cavity Armature	Eyes	Lunules IV	Granulation on Legs	Egg Type	Egg Process Height (in μm)	Egg Process Base Width (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	cone with cap	10–12
Paramacrobiotus arduus	smooth	I–III	absent	smooth	I–IV	richtersi	12.1–18.3	10.4–16.3	simple cone	16–21
Paramacrobiotus areolatus	smooth	I–III	present	crenate	I–IV	areolatus	20.0–28.0	19.0–22.0	simple cone	?
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	simple cone (jagged)	15–19
Paramacrobiotus centesimus	smooth	I–III	absent	smooth	I–IV	areolatus	7.0–11.0	?	simple cone	11–12
Paramacrobiotus chieregoi	smooth	I–III	absent	smooth	?	chieregoi	?	?	elongated cone	14
Paramacrobiotus corgatensis	sculptured	I–III	present	dentate	?	richtersi	20.0–25.0	18.0–24.0	simple cone (jagged)	8–11
Paramacrobiotus csotiensis	smooth	II–III	present	?	?	csotiensis	?	?	hemispherical covered with a hyaline layer	?
Paramacrobiotus danielae	sculptured	I–III	present	smooth	?	areolatus	14.5	24.7	simple cone	?
Paramacrobiotus danielisae	sculptured	I–III	absent	smooth	?	richtersi	17.3–23.0	17.5–20.0	simple cone	9–10
Paramacrobiotus depressus	smooth	I–III	absent	smooth	IV	richtersi	9.3–12.4		simple cone	16–23
								12.4–15.2
Paramacrobiotus derkai	smooth	I–III	present	smooth	I–IV	huziori	8.0–17.1	12.5–28.3	simple cone	12–16
Paramacrobiotus experimentalis	smooth	I–III	absent	smooth	IV	areolatus	10.3–14.9	13.8–19.4	simple cone	10–12
Paramacrobiotus fairbanksi	smooth	I–III	absent	smooth	I–IV	richtersi	10.9–14.9	10.9–20.8	simple cone (jagged)	?
Paramacrobiotus filipi	granulation	I–III	absent	smooth	I–IV	richtersi	17.8–25.2	11.7–21.7	cone with cap	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	cone with cap	10–13
Paramacrobiotus gerlachae	smooth	I–III	absent	smooth	IV	richtersi	11.8–14.5	16.8–18.7	simple cone	?
Paramacrobiotus halei	sculptured	I–III	absent	?	I–IV	richtersi	11.0–14.0	22.0–23.5	blunt cone	11
Paramacrobiotus hapukuensis	smooth	I–III	absent	smooth	absent	–areolatus	15.7–21.1	14.8–16.6	elongated cone	10
Paramacrobiotus huziori	smooth	I–III	present	smooth	I–IV	huziori	20.0–33.0	20.0–30.0	simple cone	9–11
Paramacrobiotus intii	smooth	II–III	present	dentate	I–IV	areolatus	15.4–24.4	22.0–34.0	simple cone	9–10
Paramacrobiotus kenianus	smooth	?	present	?	?	richtersi	13.5 ± 1.9	19.7 ± 2.7	simple cone	17.7 ± 3.6
Paramacrobiotus klymenki	smooth	I–III	absent	dentate	I–IV	areolatus	14.5–18.5	16.4–18.2	simple cone	10–11
Paramacrobiotus lachowskae	smooth	I–III	present	smooth	I–IV	areolatus	17.6–32.1	8.1–17.7	hemispherical with filaments	8–14
Paramacrobiotus lorenae	smooth	I–III	absent	smooth	I–IV	richtersi	25.0–42.2	17.8–23.0	elongated cone	?
Paramacrobiotus magdalenae	smooth	I–III	present	smooth	IV	richtersi	13.0–25.0	16.2–21.0	simple cone	10–12
Paramacrobiotus metropolitanus	smooth	I–III	absent	smooth	IV	areolatus	7.4–14.6	9.8–21.1	simple cone	10–15
Paramacrobiotus palaui	smooth	?	present	?	?	richtersi	10.2 ± 1.3	13.4 ± 1.3	simple cone	15.4 ± 1.4
Paramacrobiotus peteri	smooth	I–III	absent	smooth	?	areolatus	10.0–14.0	9.0–12.0	simple cone (jagged)	?
Paramacrobiotus pius	smooth	I–III	absent	smooth	I–IV	richtersi	up to 12.3	19.5–24.7	simple cone (jagged)	10
Paramacrobiotus priviterae	smooth	I–III	present	smooth	I–IV	richtersi	11.8–15.0	12.9–16.3	simple cone (jagged)	?
Paramacrobiotus richtersi	smooth	I–III	absent	smooth	I–IV	richtersi	17.1–22.1	17.2–22.2	simple cone	13–17
Paramacrobiotus rioplatensis	smooth	I–III	present	smooth	?	areolatus	ca. 4.6	?	elongated cone	17–19
Paramacrobiotus sagani	granulation	I–III	present	smooth	I–IV	richtersi	9.4–13.2	14.6–22.4	blunt cone	10–13
Paramacrobiotus savai	smooth	I–III	present	smooth	IV	areolatus	12.0–18.0	16.7–18.5	blunt cone	?
Paramacrobiotus sklodowskae	smooth	I–III	present	smooth	I–IV	richtersi	16.0–17.5	20.5–23.5	blunt cone	10
Paramacrobiotus spatialis	smooth	I–III	absent	smooth	I–IV	richtersi	13–16	15.2–20.4	simple cone	15–23
Paramacrobiotus spinosus	smooth	I–III	absent	smooth	I–IV	richtersi	22.1–42.2	17.3–26.0	elongated cone (jagged)	10–11
Paramacrobiotus submorulatus	smooth	II–III	present	?	?	submorulatus	7.0–8.3	17.5–20.4	hemispherical with concave on top	13
Paramacrobiotus tonollii	smooth	?	present	smooth	?	tonollii	32.0–35.0	?	elongated cone	8–10
Paramacrobiotus vanescens	faint punctuation	I–III	absent	?	I–IV	richtersi	16.0–17.0	24.0–25.0	blunt cone (jagged)	9–12
Paramacrobiotus walteri	smooth	I–III	present	dentate	I–IV	areolatus	10.0–17.0	9.0–20.0	simple cone (jagged)	?
Paramacrobiotus wauensis	smooth	I–III	absent	?	?	?	?	?	?	?

Note: I–IV represents the number of pair of legs and ? means unsuitable or not present.

Table 1. Selected morphological characters of the known species of genus Paramacrobiotus (schematic illustrations of different types of egg process shapes presented in Figure 1).

Species	Cuticle	Number of Rows in Oral Cavity Armature	Eyes	Lunules IV	Granulation on Legs	Egg Type	Egg Process Height (in μm)	Egg Process Base Width (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	cone with cap	10–12
Paramacrobiotus arduus	smooth	I–III	absent	smooth	I–IV	richtersi	12.1–18.3	10.4–16.3	simple cone	16–21
Paramacrobiotus areolatus	smooth	I–III	present	crenate	I–IV	areolatus	20.0–28.0	19.0–22.0	simple cone	?
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	simple cone (jagged)	15–19
Paramacrobiotus centesimus	smooth	I–III	absent	smooth	I–IV	areolatus	7.0–11.0	?	simple cone	11–12
Paramacrobiotus chieregoi	smooth	I–III	absent	smooth	?	chieregoi	?	?	elongated cone	14
Paramacrobiotus corgatensis	sculptured	I–III	present	dentate	?	richtersi	20.0–25.0	18.0–24.0	simple cone (jagged)	8–11
Paramacrobiotus csotiensis	smooth	II–III	present	?	?	csotiensis	?	?	hemispherical covered with a hyaline layer	?
Paramacrobiotus danielae	sculptured	I–III	present	smooth	?	areolatus	14.5	24.7	simple cone	?
Paramacrobiotus danielisae	sculptured	I–III	absent	smooth	?	richtersi	17.3–23.0	17.5–20.0	simple cone	9–10
Paramacrobiotus depressus	smooth	I–III	absent	smooth	IV	richtersi	9.3–12.4		simple cone	16–23
								12.4–15.2
Paramacrobiotus derkai	smooth	I–III	present	smooth	I–IV	huziori	8.0–17.1	12.5–28.3	simple cone	12–16
Paramacrobiotus experimentalis	smooth	I–III	absent	smooth	IV	areolatus	10.3–14.9	13.8–19.4	simple cone	10–12
Paramacrobiotus fairbanksi	smooth	I–III	absent	smooth	I–IV	richtersi	10.9–14.9	10.9–20.8	simple cone (jagged)	?
Paramacrobiotus filipi	granulation	I–III	absent	smooth	I–IV	richtersi	17.8–25.2	11.7–21.7	cone with cap	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	cone with cap	10–13
Paramacrobiotus gerlachae	smooth	I–III	absent	smooth	IV	richtersi	11.8–14.5	16.8–18.7	simple cone	?
Paramacrobiotus halei	sculptured	I–III	absent	?	I–IV	richtersi	11.0–14.0	22.0–23.5	blunt cone	11
Paramacrobiotus hapukuensis	smooth	I–III	absent	smooth	absent	–areolatus	15.7–21.1	14.8–16.6	elongated cone	10
Paramacrobiotus huziori	smooth	I–III	present	smooth	I–IV	huziori	20.0–33.0	20.0–30.0	simple cone	9–11
Paramacrobiotus intii	smooth	II–III	present	dentate	I–IV	areolatus	15.4–24.4	22.0–34.0	simple cone	9–10
Paramacrobiotus kenianus	smooth	?	present	?	?	richtersi	13.5 ± 1.9	19.7 ± 2.7	simple cone	17.7 ± 3.6
Paramacrobiotus klymenki	smooth	I–III	absent	dentate	I–IV	areolatus	14.5–18.5	16.4–18.2	simple cone	10–11
Paramacrobiotus lachowskae	smooth	I–III	present	smooth	I–IV	areolatus	17.6–32.1	8.1–17.7	hemispherical with filaments	8–14
Paramacrobiotus lorenae	smooth	I–III	absent	smooth	I–IV	richtersi	25.0–42.2	17.8–23.0	elongated cone	?
Paramacrobiotus magdalenae	smooth	I–III	present	smooth	IV	richtersi	13.0–25.0	16.2–21.0	simple cone	10–12
Paramacrobiotus metropolitanus	smooth	I–III	absent	smooth	IV	areolatus	7.4–14.6	9.8–21.1	simple cone	10–15
Paramacrobiotus palaui	smooth	?	present	?	?	richtersi	10.2 ± 1.3	13.4 ± 1.3	simple cone	15.4 ± 1.4
Paramacrobiotus peteri	smooth	I–III	absent	smooth	?	areolatus	10.0–14.0	9.0–12.0	simple cone (jagged)	?
Paramacrobiotus pius	smooth	I–III	absent	smooth	I–IV	richtersi	up to 12.3	19.5–24.7	simple cone (jagged)	10
Paramacrobiotus priviterae	smooth	I–III	present	smooth	I–IV	richtersi	11.8–15.0	12.9–16.3	simple cone (jagged)	?
Paramacrobiotus richtersi	smooth	I–III	absent	smooth	I–IV	richtersi	17.1–22.1	17.2–22.2	simple cone	13–17
Paramacrobiotus rioplatensis	smooth	I–III	present	smooth	?	areolatus	ca. 4.6	?	elongated cone	17–19
Paramacrobiotus sagani	granulation	I–III	present	smooth	I–IV	richtersi	9.4–13.2	14.6–22.4	blunt cone	10–13
Paramacrobiotus savai	smooth	I–III	present	smooth	IV	areolatus	12.0–18.0	16.7–18.5	blunt cone	?
Paramacrobiotus sklodowskae	smooth	I–III	present	smooth	I–IV	richtersi	16.0–17.5	20.5–23.5	blunt cone	10
Paramacrobiotus spatialis	smooth	I–III	absent	smooth	I–IV	richtersi	13–16	15.2–20.4	simple cone	15–23
Paramacrobiotus spinosus	smooth	I–III	absent	smooth	I–IV	richtersi	22.1–42.2	17.3–26.0	elongated cone (jagged)	10–11
Paramacrobiotus submorulatus	smooth	II–III	present	?	?	submorulatus	7.0–8.3	17.5–20.4	hemispherical with concave on top	13
Paramacrobiotus tonollii	smooth	?	present	smooth	?	tonollii	32.0–35.0	?	elongated cone	8–10
Paramacrobiotus vanescens	faint punctuation	I–III	absent	?	I–IV	richtersi	16.0–17.0	24.0–25.0	blunt cone (jagged)	9–12
Paramacrobiotus walteri	smooth	I–III	present	dentate	I–IV	areolatus	10.0–17.0	9.0–20.0	simple cone (jagged)	?
Paramacrobiotus wauensis	smooth	I–III	absent	?	?	?	?	?	?	?

Note: I–IV represents the number of pair of legs and ? means unsuitable or not present.

3. 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 ITS-2 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., [48]). The COI mtDNA molecular marker, in particular, has been recommended for DNA barcoding purposes (http://www.barcodinglife.org accessed on 10 July 2023), such as rapid species identification, discrimination between cryptic species, and resolving phylogenetic relationships among closely related species [49,50]. 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 markers [24].

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 underestimated [11,23]. One significant challenge that needs to be addressed in future studies is the lack of available barcodes. Despite the designation of 45 species in 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. The alignment of COI barcode sequences resulted in 574 characters, with 270 variable sites and 241 parsimony informative sites. We used the COI sequence of Milnesium berladnicorum Ciobanu, Zawierucha, Moglan & Kaczmarek, 2014 [51] as the outgroup to construct the most reliable evolutionary tree. To determine the most appropriate model of sequence evolution, we applied jModelTest v. 2.1.4 [52] with both the Bayesian Information Criterion (BIC) and the Akaike Information Criterion (AIC) [53]. The GTR + G (Time-Reversible model with gamma-distributed rate heterogeneity) was selected as the best-fitting evolutionary model. The phylogenetic tree was constructed using (i) Bayesian inference (BI) analysis with the program MrBayes 3 [54], following the settings described by Mioduchowska et al. [55], and (ii) maximum likelihood (ML) analysis calculated using the program Mega X [56] with 1000 bootstraps and under the general settings of the selected evolutionary model. Uncorrected pairwise distances (p-distances) were calculated using MEGA X [56].

The binary model of phylogenetic relationships, which involves reconstructing gene trees from sequence data, allows us to gain insights into the speciation history of species [57]. 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 nodes [58]. The presence of unresolved nodes in a polytomic multifurcating tree indicates a lack of a signal in the data to resolve relationships within the genus Paramacrobiotus. This observation is partially consistent with previous studies, where both groups, richtersi and areolatus, were described as polyphyletic [11,47]. 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 observed [59]. In turn, Stec et al. [47] performed a cross-strain experiment to observe the molecular taxonomy of the genus Paramacrobiotus and indicated hidden species richness. The authors concluded that the utilization of DNA barcodes may prove inadequate in fully resolving species diversity and accurately describing species within this cosmopolitan genus. Hence, both multilocus sequencing and direct experimental testing of species boundaries are required.

The genetic p-distances between the analyzed COI barcode sequences of Paramacrobiotus species ranged from 16% to 27%, indicating different species (Supplementary Materials SM.02). However, it was shown that there are very low genetic differences, i.e., a p-distance of 0.3%, between Pam. aff. richtersi from Tunisia (GenBank: MH676016) and Pam. gadabouti from Portugal (GenBank: OP394113), suggesting they belong to the same species (Supplementary Materials SM.02). 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) (Supplementary Materials SM.02). Both sequences represented Pam. experimentalis, which is also consistent with the previous study [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 (Supplementary Materials SM.02).

4. Cryptobiosis

The stage of an organism’s life known as cryptobiosis is one in which no activity is apparent [60]. Many organisms go through cryptobiosis to survive the harsh environmental conditions they encounter [61,62,63]. A few types of cryptobiosis are known i.e., 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 [60,64]. In genus Paramacrobiotus, majority of studies related to cryptobiosis are anhydrobiosis, or the absence of water, additionally, there has also been research on famine, freezing, and bet-hedging [65,66,67,68,69]. Reuner et al. [65] studied how the influence of starvation and anhydrobiosis affects the size and number of storage cells in Pam. tonollii to understand the energetic side of anhydrobiosis. Starving Pam. tonollii for seven days led to a 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, no significant changes in storage cell size or its number in Pam. tonollii were observed. Also, the mortality was checked using prolonged starvation, and Pam. tonollii reached 50% mortality after 30 days. Likewise, Rizzo et al. [66] investigated antioxidant defences (capable of counteracting reactive oxygen species (ROS)) in Pam. richtersi in both active and dehydrated states. The 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 enzymes (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 substances reactive to thiobarbituric acid) 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, Giovannini et al. [68] performed a study in which the production of reactive oxygen species and the involvement of bioprotectants during anhydrobiosis in Pam. spatialis was investigated. The study provides evidence of an increase in ROS production relative to the time spent in anhydrobiosis, which is due to oxidative stress in the animals. Using RNA interference, the involvement of bioprotectants, including those combating ROS, was assessed. As Rizzo et al. [66] concluded, the role of glutathione peroxidase in desiccation in Pam. richtersi, this gene was targeted, and what was observed was that glutathione peroxidase gene compromised survival during the drying and rehydration of Pam. spatialis. This further strengthened the evidence that glutathione reductase and catalase play important roles during desiccation and rehydration. Also, the involvement of aquaporins 3 and 10 during the rehydration of Pam. spatialis was observed. And recently, Roszkowska et al. [69] studied the length of time that different tardigrades survive in the anhydrobiotic state, including Pam. experimentalis. The study concludes that anhydrobiotic competence is dependent on habitat instead of nutritional behaviour and the time taken to return to activity after anhydrobiosis is dependent upon the length of the anhydrobiosis. It is worth noting that in 2021, the entire genome of Paramacrobiotus sp., later described as Pam. metropolitanus, was sequenced [70]. This provides an opportunity for a better understanding of the genetic basis that enables them to survive the process of anhydrobiosis. The full DNA sequence has allowed for clues regarding the phylogeny of TPS-TPP genes responsible for the production of trehalose, a substance involved in anhydrobiosis. Four years earlier, in 2017, a similar mechanism was described in the action of the TDP protein [71]. Research conducted on the species Pam. richtersi [9], among others, revealed the involvement of this compound in DNA protection during the gradual dehydration of the organism. Further studies on the genomes of tardigrades from the genus Paramacrobiotus have the potential to uncover new information that can contribute not only to understanding the unique characteristics of these organisms, but also to gaining broader insights into evolution and the adaptive plasticity of organisms in various extreme environments.

5. Distribution

Paramacrobiotus species shows worldwide distribution. However, the real distribution of the Paramacrobiotus species is unknown due to taxonomic problems, misidentifications and lack of genetic data. This is especially visible for often reported species like Pam. areolatus or Pam. richtersi. Most of the reports of these two taxa belong to a different species. Here we present a confirmed distribution (reports from type localities or with genetic confirmation) of all 45 species in the genus Paramacrobiotus to date (in Supplementary Materials SM.01 and Figure 3).

6. Feeding Behaviour

Paramacrobiotus species are omnivorous and consume a variety of organisms, including certain cyanobacteria, algae, and fungi, as well as the rotifer, nematodes, and small juvenile tardigrades. Additionally, the diets of adults and juveniles differ: 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 cells [72].

7. Life History

Life history refers to total lifespan, development, reproduction and death of an organism [73]. 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 ovipositions [74,75]. The lifespan differs from species to species in the case of tardigrades [76]. The life histories of only a few Paramacrobiotus species have been reported to date, that is, Pam. fairbanski, with an average lifespan of 137.3 ± 136.4 days and 194.9 ± 164.4 days and age at first oviposition of 70.7 ± 19.4 days and 76.9 ± 16.4 days [77]; Pam. kenianus, with an average lifespan of 125 ± 35 days and 141 ± 54 days, a maximum lifespan of 204 days and 212 days, and age at first oviposition of 10 days and 10 days [74]; Pam. metropolitanus, with juveniles hatching in 12–20 days and first oviposition within 11–13 days after hatching [78]; Pam. palaui, with an average lifespan of 97 ± 31 days, a maximum lifespan of 187 days, and age at first oviposition of 10 days [60]; Pam. richtersi, with an age at first oviposition of 64.2 ± 1.7 days [79]; and Pam. tonollii, with an average lifespan of 69.0 ± 45.1 days, a maximum lifespan of 237 days, and an age at first oviposition of 24.4 ± 4.4 days [76].

8. Microbiome

The microbiome represents the entire community of microorganisms, including fungi, protists, bacteria, archaea, as well as 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 behaviour, metabolism, development, immunity, or pathogen defence, thereby regulating the functioning of the entire organism [80,81]. Conversely, it has also been demonstrated that the host’s phylogeny [82] and diet [83] have significant impacts on the overall microbial composition. Indeed, many metazoan species appear to harbour their own specific microbiome community [48]. However, our understanding of the microbiome composition of Tardigrada, based on next-generation sequencing (NGS) methods targeting the standard 16S rRNA bacterial barcoding gene fragment, is limited to a very small number of published articles [84,85,86,87,88,89,90].

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. [84] 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. 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 mutualists [91]. 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 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, 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. [88] 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 were identified as the three most prevalent phyla among the analyzed tardigrades, including species outside the genus Paramacrobiotus. However, the focus of the study was on potential tardigrade endosymbionts, particularly OTUs from the order Rickettsiales and the genus Wolbachia. Both Rickettsiales and Wolbachia were detected in the 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 insects [92]. However, recent studies have identified infections of this bacterial endosymbiont in various freshwater invertebrate species [90,93,94]. Generally, this bacterium is transmitted vertically from mother to offspring and/or through horizontal transfer directly from the environment or between different hosts [95]. Subsequently, Wolbachia manipulates host reproduction by inducing parthenogenesis, feminization, male killing, or cytoplasmic incompatibility [96,97].

In 2023, Mioduchowska et al. [90] 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 analyzed by Kaczmarek et al. [85]. 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.

9. Reproduction

Reproduction refers to the process whereby every known organism produces offspring either sexually or asexually. In the 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 (a form of asexual reproduction when only females are present in the population) [98]. The genus Paramacrobiotus consists of both bisexual and unisexual species/populations. The Pam. arduus from Italy is bisexual; the Pam. areolatus population from Italy is bisexual; the population from Svalbard is unisexual; Pam. celsus from Italy is bisexual; Pam. depressus from Italy is bisexual; Pam. experimentalis from Madagascar is bisexual; Pam. fairbanksi from various locations such as the Antarctic, Italy, Poland, Spain and USA is unisexual; Pam. filipi from Borneo is unisexual; Pam. gadabouti from various locations in Portugal, Australia, France and Tunisia is unisexual; Pam. kenianus from Kenya is unisexual; Pam. metropolitanus from Japan is bisexual; Pam. palaui from Micronesia is unisexual; Pam. richtersi from Ireland is bisexual, and according, to modern taxonomy, probably constitutes a distinct species; Pam. spatialis from Italy is bisexual; and Pam. tonolli from the USA is bisexual. Out of 45, the mode of reproduction for only 14 species is known (Supplementary Materials SM.01).

An important aspect of reproduction is the morphology of sperm, the types of fertilization and reproductive strategies. In bisexual Paramacrobiotus species, external fertilization has been observed, which occurs after the female lays eggs [9]. Sperm in this group of tardigrades are characterized by a longer acrosome compared to genera like Mesobiotus, Xerobiotus or Macrobiotus [32]. A similar situation occurs in the case of the tail. The size in the genus Paramacrobiotus ranges from 13 μm to 29.4 μm, which is considerably longer than in the genus Macrobiotus (9.4–24.2 μm) [99]. Such dimensions are crucial when discussing the speed of movement of male gametes, which increases with tail length [100]. However, the length of the tail can change when the sperm enters the spermatheca. In the species Paramacrobiotus sp. and Macrobiotus shonaicus (Stec, Arakwa & Michalczyk 2018) [101], such changes were observed for the first time, characterized by a shortening of the tail (1.3–3.6 μm). This reduction is natural, as once the sperm reaches the spermatheca, the tail ceases to serve its purpose, and its length becomes nonessential [99]. Within species, there are often many differences in sperm morphology (length of the nucleus, acrosome and tail), which can be potentially useful in the context of research on the taxonomy of the genus Paramacrobiotus [32].

Among the species in this genus, a significant correlation between reproductive strategy and karyotype has been observed [9]. For example, in different populations (from Ireland and Italy) of Pam. richtersi, different chromosomal compositions within the COX1 gene were found. It was observed that in the population consisting of only females (an apomictic phenomenon), animals were triploid, and they underwent ameiotic oocyte maturation. In the case of the bisexual species, individuals were diploid, with chromosomal pairing occurring during oocyte and spermatocyte maturation [9]. These observed reproductive differences, genetic studies, and variations in egg morphology allowed the distinction of four new taxa within the Pam. richtersi species complex.

Also, Guidetti et al. [11] suggest the mode of reproduction being related to a constrained or wide distribution of the species. The amphimictic species display 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.

10. Key for Species Identification

11. Conclusions

The genus Paramacrobiotus shows a cosmopolitan distribution with the presence of both bisexual and parthenogenetic species. Although the integrative descriptions and redescriptions are improving the overall situation and allowing for new opportunities for detailed study, the phylogeny of the genus Paramacrobiotus seems to be unresolved. Also, there are many other studies regarding the 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 knowledge of tardigrades in general.