A MORPHOLOGICAL AND BIOLOGICAL ASSESSMENT OF CRENOBIA ALPINA AND CRENOBIA MONTENIGRINA (PLATYHELMINTHES, TRICLADIDA, PLANARIIDAE) IN THE PARÂNG MOUNTAINS (ROMANIA)

The paper analyses the morphology of the copulatory apparatus and the reproductive isolation of the monopharyngeal and polypharyngeal Crenobia (Dana, 1766) in sympatric populations of the Parâng Mountains, Romania. The copulatory apparatus of the two forms shows a non-simultaneous development, indicating a seasonal reproductive isolation: the monopharyngeal Crenobia alpina (Dana, 1766) becomes able to mate during the warm season while the polypharyngeal Crenobia montenigrina (Mrázek, 1904) becomes able to mate during the cold season. The mature copulatory apparatus of the monopharyngeals and the polypharyngeals shows all the characters typical for C. alpina , respectively for C. montenigrina as presented in the literature. In both species, some specimens reveal a complex system of fine ducts / sclerotized lines into the wall of the genital atrium, which are attributed to biological structures of unknown function rather than histological artefacts.


Introduction
Crenobia is a genus of freshwater flatworm with a limited dispersal ability which lives almost exclusively in cold springs and headwaters of mountainous areas in Europe and Turkey (Brändle et al. 2007, Brändle et al. 2017, Sluys 2022).
Taxonomically, Crenobia is a group of species (Crenobia alpina sensu lato) with different morphology, reproductive biology, karyology, ecological requirements, habitat, geographic distribution (Sluys 2022).The various forms of this complex were regarded either as distinct species within the genus Crenobia or as subspecies of Crenobia alpina (see the synthetic review of Sluys, 2022).
The forms present in the Romanian fauna are the monopharyngeal C. alpina and the polypharyngeal C. montenigrina (Babalean 2020, Codreanu 1956, Sluys 2022).In the Parâng Mountains the two forms occur sympatrically in mixed populations (Babalean 2020).Codreanu (1956) indicates the seasonal reproductive isolation of C. alpina and C. montenigrina.The copulatory apparatus of the two forms is very similar; the minor differences between them should not prevent mating.The two morphological types are discussed from the biological species concept, of reproductive isolation, according to literature (Bănărescu 1973, De Queiroz 2007).
The aim of the paper is to assess and verify the seasonal reproductive isolation of the monopharyngeals and polypharyngeals, on morphological grounds.

Types of populations
In the studied area, C. alpina and C. montenigrina formed the following types of populations with respect to the pharyngeal condition (mono-/ polypharyngeal) and the number of collected specimens: A) non-mixed populations a) exclusively monopharyngeals, with a small number of specimensonly one monopharyngeal population (ski lift area) b) large populations, exclusively polypharyngealssprings and some collecting streams B) mixed populations: a) predominant monopharyngealscollecting stream at the Paltinul Mountain base b) predominant polypharyngealssome collecting streams c) nearly equal proportions of mono-and polypahryngealsreocrene no. 3 (CB-Romanul) Some non-mixed populations were identified as mixed at the next collection (next years) and in the different seasons (warm seasoncold season) -reocrene no. 2 (CB-Romanul).However, the existence of non-mixed populations exclusively monopharyngeals or exclusively polypharingeals is uncertain because no population was sampled completely.

Morphology
The monopharyngeal Crenobia alpina (specimens CR1, Cm1, Cr4, Cm2) These four specimens were collected in summer and are sexually mature.The mouth opening is located at the posterior end of the pharyngeal pocket.With very few exceptions these specimens reveal ovaries, few prepharyngeal ventral follicular testes, ducts and fully developed copulatory apparatus.The exceptions are attributed to a physiological state (the lack of ovaries and oviducts in specimen CR1 is considered a physiological state / a stage of the reproductive cycle).The copulatory apparatus (Figs.2-6).
The copulatory apparatus match the patterns of the genus Crenobia and C. alpina: -an oversized (well developed) atrium consisting of 2 distinct parts: (i) the external muscular part, very thick, composed by muscular plates (bundles) in radial disposition, surrounded by an outer zone of myoblasts (Sluys 2022) and (ii) the internal fibrous layer -the penis has an indistinct bulb and a long papilla, extending beyond the atrial fold -Fig.3 -the genital atrium is divided by an atrial fold in 2 regions: (i) the proximal region which houses the most part of the penis papilla; it also receives the common oviduct and the bursal canal (Sluys 2022) and (ii) a distal region which opens by the gonopore (the genital orifice).
-the two vasa deferentia (spermiducts) with large spermiducal vesicles in their terminal part, at a certain distance before opening into the penis bulb.
Besides the above general characters, the histological slides of the four analysed specimens reveal some peculiarities: the course of the two vasa deferentia is symmetric in specimens CR1 and CR4 and asymmetric in specimen Cm1; the presence of a complex system of apparently fine ducts or sclerotized lines in the wall of the genital atrium (Figs.2-4).These structures seem to be arranged between the atrial space housing the penis papilla, the distal atrium, and the penis bulb, interconnecting them.Orifices corresponding to the fine ducts are visible in the penis bulb and in the wall of the distal atrium.Besides the pigmented specimens, unpigmented (white) and marbled polypharyngeals were also collected (Fig. S1).Numerous pharynxes, opening into a common space with a posterior mouth, at a certain distance from the genital orifice (shifted anteriad) (Fig. 7).
The mature specimen CR8 show small and rare pre-pharyngeal follicular testes located ventrally.The copulatory apparatus (Figs. 7, 8) presents most of the characteristics of C. montenigrina as described in the literature (Sluys 2022), excepting the asymmetry of the vasa deferentia.The fine ducts / sclerotized lines are present but to a much lesser extent than in the monopharyngeals.The immature polypharyngeal specimens (sampled in summer) reveal incompletely developed copulatory apparatus, developing follicular testes and ovaries on histological sections (Fig. S2).
Morphologically, the Romanian specimens of both Crenobia alpina and Crenobia montenigrina fit quite well the description from literature (Sluys 2022).Minor differences such the symmetry of the vasa deferens in specimen CR1 (monopharyngeal) and the asymmetry in specimen CR8 (polypharyngeal) might be individual characters (intrapopulational variability).
A comparison of the morphological characters analysed in this paper for the two species is presented in Table 1 Penis papilla Extends beyond the atrial constriction -Fig.3 Extends beyond the atrial constriction -Fig.7 Table 1.A synthetic comparison between C. alpina s. str.and C. montenigrina The characters with diagnostic value appear to be the number of pharynxes and the position of the mouth opening; the two species appear to be cryptic at the level of the copulatory apparatus.
The complex system of fine ducts / sclerotized lines are possibly genuine biological elements involved for instance in shedding and releasing cocoons.

Aspects regarding the reproductive biology (Figs. 9, 10; Table 2) The monopharyngeal Crenobia alpina
During the warm season (June, July), the following types of monopharyngeals were collected: mostly specimens with large, well-developed copulatory area (Fig. 9Aa); two specimens with a dilated, oversized copulatory area having the aspect of an internal cocoon (Fig. 9 Ab); a few specimens with small copulatory area; a few asexual specimens, without copulatory area.External cocoons were not observed.
During the cold season (November), most part of the monopharyngeals show small copulatory area, a smaller proportion are asexual without copulatory area.The asexuals have a distinct rounded appearance of the posterior end (Fig. 9B).The rounded posterior end is here associated with hatching, such specimens resulted by hatching.Fission not observed.Mating not observed.

The polypharyngeal Crenobia montenigrina
During the warm season (June, July) most specimens have small copulatory area, and some are asexual (they lack the copulatory area).
During the cold season (November), all the specimens in most springs presented large copulatory area; in only one investigated spring, all specimens were asexuals with no copulatory areareocrene no. 2 (CB-Romanul), November 2022.
Neither external, nor internal cocoons were observed.The polypharyngeals were found undergoing fission during July and November in non-mixed populations (for instance reocrene spring no. 2 -July 2021, Păpușa spring -November 2020 (Fig. 10 A, B).Specimens most probably resulted from fission were sampled in November (Tidvele area); their rear end has a tapering appearance (Fig. 10C).
The population of the reocrene spring no. 2 (CB-Romanul) consisted of only immatures polypharyngeals on 05 July 2021, and as a mixture of 7 asexual polypharyngeals and one asexual monopharyngeal on 07 November 2022.In this latter case of mixed population, polypharyngeals and monopharyngeals cannot interbreed due to their immaturity.This spring was found almost completely deteriorated in November 2022.Mating not observed in polypharyngeals.The monopharyngeals mate and produce cocoons (internal, maybe external as well) during the warm season (June, July).The few asexuals or those with small copulatory area are most probably resulted from cocoons hatching.These hatchlings grow and start to develop the copulatory area.They become specimens with small copulatory area, unable for sperm transfer in the cold season (November).The November asexuals (with no copulatory area) result from late summer /early autumn hatching.
The polypharyngeals mate during the cold season when they have completely developed copulatory area.They are unable to mate during the warm season, as they have small copulatory area or lack it.
This study strongly indicates the seasonal reproductive isolation of C. alpina and C. montenigrina.A deeper and true reproductive isolation should be verified by the comparative study of spermatozoa in the two forms.
The natural history of C. alpina and C. montenigrina, much discussed in the literature (Codreanu 1956 and reference therein) remains still unsolved and raises many questions: When and how the two species appeared?Why do they form sympatric populations in the Carpathian (Parâng) Mountains?Are they sympatric elsewhere in Europe?As to their origin, three possibilities can be discussed: 1) C. alpina and C. montenigrina have a common ancestor which disappeared from the current fauna.In this hypothesis, from a common pre-glacial ancestor (before the Quaternary Glaciation) resulted sympatric C. alpina and C. montenigrina -with a much northern geographical distribution, later modified by the glaciation.Both species are cryophilic, but regarding the reproductive period, C. montenegrina is more cryophilic (sexually mature in the cold season).In the cold periods of the Glaciation (the glaciation stadials) both species would have undergone a southward migration.C. montenigrina settled in high mountainous areas, corresponding to their thermal optimum.In warm periods (interstadials), C. alpina would have undergone the reverse migration path from south to north while C. montenigrina did not.2) C. montenigrina resulted from C. alpina.This theory must admit the present geographical range of C. montenigrina as speciation center.A question that needs to be answered is: How the pharyngeal number (one / numerous) is related to shifting reproductive season?3) C. alpina resulted from C. montenigrinafor the moment this hypothesis is difficult to be accepted.

Conclusions
The present results are consistent with the theory of temporal reproductive isolation of the two species, expressed by Codreanu (1956).C. alpina and C. montenigrina remain species of great interest for future studies.The factors that influence the reproductive biology of the two species in sympatry are poorly understood.

Figure 9 .
Figure 9. Crenobia alpina, general habitus: Ain summer, alarge copulatory area, boversized copulatory area; Bin November, small asexual specimen with rounded posterior end, most probably resulted from the late summer /early autumn hatching.