The Contribution of Historical and Morphological Studies on Herbarium Specimens to a Better Definition of Chara pelosiana Avetta (Charales, Charophyceae)

The lectotype of Chara pelosiana Avetta 1898 was designated in 2000 by Langangen, who merged the species with Chara fibrosa Agardh ex Bruzelius. Chara pelosiana belongs to the section Agardhia Wood, but the true identity of the species has yet to be confirmed. The purpose of this work is to show some historical and morphological findings regarding this enigmatic species, on the basis of the analysis of herbarium specimens. The original material, which was studied by Avetta, is missing in Italian herbaria, but portions of it have been found in the Herbarium of Jena. Historical research on botanists related with this species resulted in the discovery of several specimens to be considered “original material”, and new unpublished localities in Northern Italy. Morphological observations have been made on portions of herbarium specimens as a contribution to unveil the taxonomic identity of this taxon. The specimens are diplostichous with ecorticate branchlets, have stipulodes in a single row, one or two per branchlet, and spine cell up to 1 mm long.


Introduction
Chara pelosiana was published in 1898 by Avetta [1] upon a specimen with a single row of stipulodes and ecorticate branchlets that was collected in 1886 in rice fields in S. Anna (near S. Cesario, Province of Modena, Northern Italy). C. pelosiana is one of the rarest haplostephanous species in Europe [1] (p. 229).
The species was part of Enrico Ferrari's "small but interesting collection of Characeae" [1] (p. 230), collected for the University of Rome's Botanical Institute, and was first studied by Alpinolo Pelosi, a young Natural Sciences student who died prematurely in 1887. Following Pelosi's death, the Ferrari collection, as well as the few notes and observations left by Pelosi, were gathered by Carlo Avetta and stored in Parma [1] (p. 230). However, both the Rome and Parma Herbaria have since lost track of Ferrari's collection and Pelosi's documentation.
Pelosi identified the specimen as a variety of Chara scoparia Bauer (actually Chara baueri A. Braun) [1] (p. 234). When Avetta examined it, he noticed that the cortex was diplostichous rather than triplostichous, as it is in C. baueri, and assumed he was dealing with a new species [1] (p. 232). As a result, in honor of Pelosi, he named the species Chara pelosiana.
We started looking for Ferrari's collection in 2009. A C. pelosiana specimen collected from S. Anna was discovered at the University of Turin Herbarium [2].
C. pelosiana has only been mentioned once in the Italian literature since Avetta's publication [3] (p. 16). For nearly all of the twentieth century, there was no further record of the species in Italy. Plants that looked like C. pelosiana were discovered in 1999 in rice fields in the Province of Ferrara (Northern Italy) [4]. They were named C. fibrosa Agardh ex Bruzelius ssp. benthamii (A. Braun) Zaneveld, following Soulié-Märsche et al. [5].
It was Langangen who merged C. pelosiana with C. fibrosa, choosing fragments of the species collected in S. Anna and housed in the Herbarium of Oslo to be the lectotype of The historical search for C. pelosiana was based on an examination of the limited available literature [1][2][3] and, more importantly, a study of the herbarium materials kept in JE, LD, MOD, PAD, PARMA, PAV, RO, and TO (herbarium acronyms according to [16]). All of these Herbaria are related to botanists who have investigated or collected this species (see Appendix A). Further requests were sent without success to the Italian Herbaria BOLO, CAT, FI, NAP, PAL, and PI, which preserve historical collections of algae.
Furthermore, manuscript documents found attached to the specimens, as well as a selection of letters kept in the Archive of Botanical Garden of the University of Padua [17] provided significant additional information.
For the morphological investigation, portions of C. pelosiana from the Herbaria of MOD, TO, and PAV, as well as from the specimens kept in PAD Herbarium, were taken and transferred to Rome for examination and photography. The fragments stored in JE were insufficient for portions to be removed for further study. Morphological observations were made using a Zeiss stereomicroscope equipped with a Leica DFC 42 digital camera. The material was photographed either dry or after being rehydrated and decalcified using a 1N hydrochloric acid solution.

Historical Findings
Unfortunately, neither RO nor PARMA, where Carlo Avetta worked from 1893 until his retirement, kept the original collection. Nevertheless, C. pelosiana specimens from the original site and other Italian locations have been discovered in several Italian and foreign Herbaria.
Based on the importance of the exsiccata, the C. pelosiana specimens were divided into three groups. The data labels for each specimen were faithfully returned and noted.

Herbaria That Keep Original Material of the Name Chara pelosiana
Jena Herbarium (JE): Small fragments of the species established by Avetta are kept in an envelope with the stamp "Herbarium Walter Migula Eisenach" in the top right corner. On the envelope, Avetta wrote "Chara Pelosiana Avetta" (Figure 1A,B).
A postcard from Avetta to Migula, dated November 9, 1898, is attached to the sample, and reports, in French, "Mr. le Prof. Migula | Parma 9-11-98 | Je vous envoye un tout petit échantillon d'une Chara italienne que je viens de décrire comme espèce nouvelle (Malpighia dernière livraison) et au sujet de laquelle je voudrais bien connaitre votre opinion, quelconque elle soit ( . . . ) Dr. C. Avetta | Jardin botanique-Parma" [( . . . ) I am sending you a very small sample of an Italian Chara that I just described as a new species (Malpighia last issue) and would like to hear your opinion on it, whatever it is ( . . . )] ( Figure 1C). am sending you a very small sample of an Italian Chara that I just described as a new species (Malpighia last issue) and would like to hear your opinion on it, whatever it is (…)] ( Figure 1C). Notes. The fragments are extremely significant because they come from the original material examined by Avetta, although the labels do not mention any locality. According to a footnote in Avetta's paper, they were sent to Migula to obtain his opinion on the validity of the new species [1] (p. 229). In the same footnote, Avetta reports that the new taxon was published without confirmation from Migula, who was away for a few months and could not examine the specimen. As a result, the publication does not provide an illustration of the new species. Avetta goes on to say that the figures will appear in his next note on the Italian Characeae, which, however, was never published.
Modena Herbarium (MOD): The specimen consists of six small fragments pinned to a herbarium sheet (Figure 2A) Figure 2B).
The preprint revision label was written by Leone Formiggini, who was engaged in revisionary work on the Italian Characeae with Augusto Béguinot (see Appendix A). The label is free within the folder, with the name of the research project, Characeae Italicae, at the top and the institution where the project was located, Patavii, ex R. Instituto botanico, at Notes. The fragments are extremely significant because they come from the original material examined by Avetta, although the labels do not mention any locality. According to a footnote in Avetta's paper, they were sent to Migula to obtain his opinion on the validity of the new species [1] (p. 229). In the same footnote, Avetta reports that the new taxon was published without confirmation from Migula, who was away for a few months and could not examine the specimen. As a result, the publication does not provide an illustration of the new species. Avetta goes on to say that the figures will appear in his next note on the Italian Characeae, which, however, was never published.
Modena Herbarium (MOD): The specimen consists of six small fragments pinned to a herbarium sheet (Figure 2A) Figure 2B). Notes. The first duplicate of Ferrari's original set collected in rice fields in the province of Modena is kept in MOD. When the Botanical Institute of Rome requested a collection of Characeae from this area, Ferrari was still working at the University of Modena (see Appendix A). Unfortunately, no Ferrari autograph labels can be seen in MOD.
The Ferrari Characeae collection consists of 26 specimens of different species collected near Modena between 1878 and 1886, 14 of which were collected in 1886 (Table 1). All specimens have Modena Herbarium preprint labels with the stamp "Hortus Reg. Botanicus Mutinensis". The collection is not numbered, there is no indication of Legit, and all of the labels were handwritten by two unidentified people. The homogeneity of the compilation becomes apparent when comparing the first handwriting, which included the binomial, locality, and date, as if the labels were filled in all at once by an amanuensis, rather than by a botanist. The second anonymous handwriting only provided information about who made identification, in this case, an Institute, the Botanical Institute of Rome.
Finally, it should be noted that Formiggini and Béguinot disagreed regarding the correct position of C. pelosiana, which, according to Béguinot, could be Lychnothamus pelosianus (see also below, the letter from Formiggini to Migula kept at JE, and the discussion). Notes. The first duplicate of Ferrari's original set collected in rice fields in the province of Modena is kept in MOD. When the Botanical Institute of Rome requested a collection of Characeae from this area, Ferrari was still working at the University of Modena (see Appendix A). Unfortunately, no Ferrari autograph labels can be seen in MOD.
The Ferrari Characeae collection consists of 26 specimens of different species collected near Modena between 1878 and 1886, 14 of which were collected in 1886 (Table 1). All specimens have Modena Herbarium preprint labels with the stamp "Hortus Reg. Botanicus Mutinensis". The collection is not numbered, there is no indication of Legit, and all of the labels were handwritten by two unidentified people. The homogeneity of the compilation becomes apparent when comparing the first handwriting, which included the binomial, locality, and date, as if the labels were filled in all at once by an amanuensis, rather than by a botanist. The second anonymous handwriting only provided information about who made identification, in this case, an Institute, the Botanical Institute of Rome.
Finally, it should be noted that Formiggini and Béguinot disagreed regarding the correct position of C. pelosiana, which, according to Béguinot, could be Lychnothamus pelosianus (see also below, the letter from Formiggini to Migula kept at JE, and the discussion).  Figure 2B].

Turin Herbarium (TO):
The specimen, which is nearly entirely fragmented, was found free within a folder with three labels. The first two labels, which are pinned to the herbarium sheet and almost joined by a third pin to form a single label, were handwritten by Ferrari (    (Table 2). TO contains 19 additional Characeae specimens that were collected by Ferrari between 1887 and 1905, mainly from Piedmont and Valle d'Aosta.  Chara hispida L. var microphylla Schumach. -- The C. pelosiana specimen kept in TO is particularly valuable because the labels were handwritten by Ferrari, the original collector, and refer to both specimen No. 3 and the type locality, S. Anna [1] (p. 234).
TO also preserves two further specimens collected by Ferrari at S. Anna on September 19, 1899 (identified by Formiggini as C. foetida A. Br. and C. fragilis Desv. f. subinermis β Hedwigii Ag.), one year after Avetta's publication and thirteen years after the first collection, suggesting that Ferrari tried unsuccessfully to again find C. pelosiana.

Oslo Herbarium (O):
The lectotype of C. pelosiana was designated by Langangen and is kept in the Oslo Herbarium [6]. The specimen, which is kept in an envelope, was Notes. Langangen did not mention how the C. pelosiana specimen reached the Oslo Herbarium. He only recalled that Nordstedt, a charophyte authority at the time, was in close contact with the phycologist N. Wille from Kristiania (Oslo).
We also searched for C. pelosiana in the Lund Herbarium (LD), which houses the original Nordstedt herbarium, but found nothing.  Notes. Langangen did not mention how the C. pelosiana specimen reached the Oslo Herbarium. He only recalled that Nordstedt, a charophyte authority at the time, was in close contact with the phycologist N. Wille from Kristiania (Oslo).
We also searched for C. pelosiana in the Lund Herbarium (LD), which houses the original Nordstedt herbarium, but found nothing. Notes. This is the second unpublished specimen of C. pelosiana. It was collected by Ferrari from the same area in the province of Modena, the Nonantola rice fields, a week before the specimen of S. Anna was collected.
Pavia Herbarium (PAV): A large amount of material is kept free in a folder with a free label written in pencil: "Risaje Campo maggiore | 16/8/86 | Traverso e Kruch" [unknown person scripsit] ( Figure 5A). Within the folder, there is also a free preprint revision label reporting, "Chara Pelosiana Avetta, determinavit D r Leone Formiggini, Giugno  Notes. This is the second unpublished specimen of C. pelosiana. It was collected by Ferrari from the same area in the province of Modena, the Nonantola rice fields, a week before the specimen of S. Anna was collected.  Instead, there are numerous fragments from PAV ( Figure 7A). They are kept in an envelope with Formiggini's preprint label pinned to it: "Chara Pelosiana Avetta | ex herbario Ticinensis | determinavit D r Leone Formiggini Giugno [June] 1907" (Figure 7B).
Notes. The information on the two envelopes indicates that Béguinot and For-  Figure 8A). Migula's revision is also written in pencil outside the envelope. It reports: "Ch. Pelosiana Avetta"with the stamp "Herbarium Walter Migula Eisenach".
A letter from Leone Formiggini to Walter Migula, still in its original envelope, is attached to the specimens ( Figure 8C). The letter is written in Italian and dated 9 July 1907: "Le invio (…) frammenti di due Caracee tratte l'una dall'erbario del R. Istituto Botanico di Modena, l'altra dall'erbario del R. Istituto Botanico di Pavia. La prima corrisponde esattamente oltre a tutto anche per località di raccolta e per data colla specie nuova descritta dal Prof. Avetta sotto il nome di Chara Pelosiana, la cui posizione sistematica sarebbe fra la Ch. Coronata e la Ch. Scoparia. La seconda è pure precisa alla precedente pure essendo raccolta in località diversa. A me sembra che questa sia sì nuova, ma vada avvicinata piuttosto al Lychnothamnus e posta in seguito al Lychn. barbatus. Infatti del Lychnothamnus ha tutto l'aspetto, solo appare come un piccolo Lychnothamnus munito di numerose ed assai lunghe spine, oltre che di un completo rivestimento corticale. (…)" "[I am sending you (…) fragments of two Italian Characeae, one from the Herbarium of the Royal Botanical Institute of Modena and the other from the Herbarium of the Royal Botanical Institute of Pavia. The first corresponds exactly, for locality of collection and date, to the new species described by Prof. Avetta under the name Chara Pelosiana, and its systematic position would be between Ch. Coronata and Ch. Scoparia. The second is very similar to the previous one even though it is collected in a different locality. It seems to me that this is indeed new, but it should be approached rather to Lychnothamnus and placed after to the Lychn. barbatus. In fact, it has in all the appearance of a Lychnothamnus, but it appears as a small Lychnothamnus equipped with numerous and very long spines, as well as a complete cortical covering. (…)]. Notes. The information on the two envelopes indicates that Béguinot and Formiggini, who both worked at Padua, took samples from MOD and PAV to their Botanical Institute for further investigation.
The portion from PAV specimen was especially valuable in our search for C. pelosiana specimens.
Jena Herbarium (JE): In addition to fragments sent by Avetta, the Herbarium of Jena also conserves portions of specimens from MOD and PAV Herbaria.
MOD's portion consists of a few fragments, which are kept in a small envelope with the following indications written in pencil inside: "Chara Pelosiana Avetta | H Mutinensis" [Formiggini scripsit]. The revision by Migula is written in pencil on a label glued to the envelope reporting "II Ch. Pelosiana Avetta"with the stamp "Herbarium Walter Migula Eisenach" (Figure 8B).
PAV portion consists of several fragments, which are kept in a bigger envelope with the indications written in pencil inside as well: "ex H. Ticinensis | Ch. Pelosiana Avetta" [Formiggini scripsit] ( Figure 8A). Migula's revision is also written in pencil outside the envelope. It reports: "Ch. Pelosiana Avetta"with the stamp "Herbarium Walter Migula Eisenach".
A letter from Leone Formiggini to Walter Migula, still in its original envelope, is attached to the specimens ( Figure 8C). The letter is written in Italian and dated 9 July 1907: "Le invio ( . . . ) frammenti di due Caracee tratte l'una dall'erbario del R. Istituto Botanico di Modena, l'altra dall'erbario del R. Istituto Botanico di Pavia. La prima corrisponde esattamente oltre a tutto anche per località di raccolta e per data colla specie nuova descritta dal Prof. Avetta sotto il nome di Chara Pelosiana, la cui posizione sistematica sarebbe fra la Ch. Coronata e la Ch. Scoparia. La seconda è pure precisa alla precedente pure essendo raccolta in località diversa. A me sembra che questa sia sì nuova, ma vada avvicinata piuttosto al Lychnothamnus e posta in seguito al Lychn. barbatus. Infatti del Lychnothamnus ha tutto l'aspetto, solo appare come un piccolo Lychnothamnus munito di numerose ed assai lunghe spine, oltre che di un completo rivestimento corticale. ( . . . )" "[I am sending you ( . . . ) fragments of two Italian Characeae, one from the Herbarium of the Royal Botanical Institute of Modena and the other from the Herbarium of the Royal Botanical Institute of Pavia. The first corresponds exactly, for locality of collection and date, to the new species described by Prof. Avetta under the name Chara Pelosiana, and its systematic position would be between Ch. Coronata and Ch. Scoparia. The second is very similar to the previous one even though it is collected in a different locality. It seems to me that this is indeed new, but it should be approached rather to Lychnothamnus and placed after to the Lychn. barbatus. In fact, it has in all the appearance of a Lychnothamnus, but it appears as a small Lychnothamnus equipped with numerous and very long spines, as well as a complete cortical covering. ( . . . )]. Notes. According to the documentation, Formiggini submitted fragments of the two C. pelosiana specimens kept in PAD, which came from the portions removed from MOD and PAV, to Migula.

Morphological Findings
Measurements of the axes and lengths of the stipulodes were taken on the dry samples. They are summarized in Tables 3 and 4.  Notes. According to the documentation, Formiggini submitted fragments of the two C. pelosiana specimens kept in PAD, which came from the portions removed from MOD and PAV, to Migula.

Morphological Findings
Measurements of the axes and lengths of the stipulodes were taken on the dry samples. They are summarized in Tables 3 and 4. These tables show that there are no consistent differences between the herbarium samples. Their axes have similar minimum and maximum diameters. The stipulodes' lengths follow the same pattern. Additionally, preliminary phylogenetic analyses of partial chloroplast gene sequence data from the Nonantola and S. Anna collections stored in TO supports their con-specificity (Kenneth G. Karol, pers. comm.).
A stereomicroscope examination of these samples also revealed that they are morphologically comparable ( Figure 9A-G and Figure 10A-E). Therefore, a single description can be extended to all the material removed.
The specimens, which are more or less heavily calcified, are almost all fragmented. Despite this, they do not appear to be longer than 7-8 cm ( Figure 10B), as reported by Avetta [1] (p. 231), who observed them closer to the time of collection than we did. The axis diameter has mean values from 383 to 514 µm (Table 3). All the axes are corticated, diplostichous, isostichous or slightly tylacanthous ( Figures 9C and 10D,E), and bear spine cells (Table 5) generally longer than the axis, sometimes in a whorl ( Figure 10E) and sometimes curved towards the axis ( Figure 9A). Stipulodes are in a single row, perpendicular to the axis, one or two per branchlet, long to 1350 µm ( Figures 9D,G and 10A,D). Branchlets are (6)8-9(10) per whorl, totally ecorticate ( Figure 10C), wide approximately half of the axes. They are composed of 3-4(6) segments bearing at each node, including the apical nodes, a crown of long bract cells (Figures 9G and 10C). The two bracteoles are longer than the oogonia ( Figure 9G). The mean length of the basal branchlet cells is 1.7-2.7 mm in the apical parts of MOD, TO, PAD, and PAV dry samples, and 3.5 mm in the fourth whorl of branchlets of the removed sample collected from S. Anna (TO).
In light of the information presented in Tables 1 and 2, and considering the nomenclatural changes, the C. pelosiana species found in rice fields in the Modena area in the week of 23 September to 1 October 1886 were accompanied by: C. vulgaris L., C. hispida sensu auct. nonnull., C. globularis Thuiller, C. tomentosa L., and Nitellopsis obtusa (Desv.) J. Groves.     The plants are monoecious with conjoined gametangia. The oogonia are 425-450 µm long (excluding the coronula) and 312-340 µm wide. The oospores are golden-brown, and have, in the dry material collected from S. Anna (TO), c. 350 µm in length, and 235 µm in width ( Figure 9F). The spiral turns are 7-8. The antheridia in the portions removed from the PAV specimen are 250-270 µm in diameter.
In light of the information presented in Tables 1 and 2, and considering the nomenclatural changes, the C. pelosiana species found in rice fields in the Modena area in the week of 23 September to 1 October 1886 were accompanied by: C. vulgaris L., C. hispida sensu auct. nonnull., C. globularis Thuiller, C. tomentosa L., and Nitellopsis obtusa (Desv.) J. Groves.

The Studies of C. pelosiana by Pelosi, Avetta, Formiggini, and Béguinot
Letters from the scientific correspondence received by Pier Andrea Saccardo (1845-1920), a professor of botany and prefect of the Botanical Garden of Padua [17], help to explain the interests of the Rome Botanical Institute in Italian Characeae.
In a letter dated 4 November 1886, Pietro Romualdo Pirotta, the director of the Botanical Institute of Rome, requested a loan of Characeae specimens from the Padua Herbarium. The loan was for Alpinolo Pelosi, "a talented young student" who had been working with Characeae for a year and whom Pirotta encouraged to pursue a monographic study of the Italian species. Between the end of 1886 and the beginning of the next year, a great number of Italian specimens from most of the Italian Herbaria, including Ferrari's small collection from the Modena area, were sent to Rome for this purpose [1] (p. 230) [17] (letters: 15 November 1886, 10 February 1887).
After Pelosi's untimely death in August 1887, Carlo Avetta, who was Pirotta's first assistant, was entrusted with the monograph of the Italian Characeae. Despite the difficulty presented by the large amount of material gathered in Rome and the study of a problematic group, Pirotta considered the work practically complete by the beginning of 1893 [17] (letters: 22 December 1890, 25 November 1891, 17 January 1893). However, Avetta had moved to Parma by the end of 1893, and Pirotta was forced to announce the conclusion of the study at the Botanical Institute of Rome, and he returned the loan of the Characeae of Padua [17] (letter: 12 April 1894).
Avetta was not a specialist of Characeae. The collections of the General Herbarium in RO house the only specimen of Chara collected by him. This specimen was identified by Formiggini and Béguinot (Ch. crassicaulis, Colli Astigiani, September 1886, det. Formiggini and Béguinot, sine data). Avetta's revisions and determinations of the genera Nitella, Tolypella, Lamprothamnium, and Lychnothamnus can be found in the collections of the General Herbarium and Cesati Herbarium in RO, although they are nearly always unsigned.
Avetta confirmed [1] (p. 230) that his study of Italian Characeae began after Pelosi's death (1887) while he was gathering the materials and notes left by the unlucky student as well as Ferrari's collection. Both records have disappeared from Rome, but there is a record of a payment made to Ferrari in 1886 for his Characeae collection from the Modena area in the RO Archive [18].
Avetta resumed his study of Characeae in 1898, after a period of interruption [1] (p. 230), with the help of the regional collections kept at RO (Roman Herbarium). The Register of loans of RO shows a single loan of 44 specimens of Characeae sent to Avetta in Parma in 1898 [19]. The collection was returned to RO only ten years later, at the beginning of 1908, without any revisions. The sending took place a few months before Avetta's publication on Malpighia, suggesting that Avetta still had Pelosi's documentation and Ferrari's collection with him when he left the University of Rome, perhaps as early as 1893, or that if these materials were forwarded to him later, they were sent privately.
Formiggini and Béguinot's views on C. pelosiana can be deduced from specimens kept in TO, MOD, PAV, and JE Herbaria as well as from documentation in RO Archive. While Formiggini appears to have agreed on the validity of the new species (see revised labels from MOD and PAV, June 1907), Béguinot At the beginning of 1908, shortly after Avetta's loan was returned, Formiggini and Béguinot examined the complete Characeae collection kept in RO, which consisted of 828 specimens from the three Herbaria: Roman (58), General (383), and Cesati (387). The Register of loans contains a detailed list of all species sent to them [20], revealing the absence of C. pelosiana, which was therefore no longer part of the RO collections ten years after Avetta's publication.
Despite this, Formiggini considered still C. pelosiana to be valid at the beginning of 1909, as revealed by his subsequent revisions in TO: S. Anna (December 1908) and Nonantola (January 1909). Despite not having seen the original C. pelosiana material, he confirmed the validity of the new species by examining specimens in TO and MOD. Furthermore, he also recognized the unpublished specimens kept in TO and PAV as C. pelosiana. The one of the two new specimens discovered during our research is therefore the one collected by Ferrari from Nonantola (Modena) almost a week before the S. Anna specimen was collected. The other was collected by Traverso and Kruch near Pavia just over a month before the type specimen was found. This indicates two new Italian stations for this species' distribution area.
To bolster this impression, each of the two assumed Chara species were followed by different observations by Pelosi, which were fully published by Avetta [1] (pp. 234-235).
In the absence of the material seen by Avetta and Pelosi's original notes, the examination of the specimens kept in TO and the information acquired in RO were decisive.
TO preserves Ferrari's only numbered collection, consisting of 25 specimens, 21 of which were collected between 20 September and 1 October 1886. This number appears to be correct, as Avetta, the last person to examine the Rome collection, described it as a "small collection" [1] (p. 230).
In the collections of the General Herbarium and Cesati Herbarium in RO, Pelosi numbered his collections (4 out of 14), revisions (50 out of 55), and determinations (10 out of 15), but the numbering system, which ranges from 4 to 130, is seriously lacking and the numbers are frequently repeated.
Nonetheless, based on a comparison of Ferrari's numbering in TO (up to 24) and Pelosi's numbering in TO and RO (up to 130), it is almost certain that the original material of C. pelosiana should be regarded as a single gathering: Ferrari's number 3 and Pelosi's revision, identified by number 101.

Which Is the Correct Identity of Chara pelosiana?
Langangen [6] merged Avetta's species with Chara fibrosa. Other authors have considered C. pelosiana to be C. fibrosa or a variety or form of this species [4,5,8,10].
C. pelosiana specimens found in herbaria or mentioned in literature in Italy [1,4] are all from rice fields.
In Zaneveld key [7], the essential differences between the three subspecies that this author includes in C. fibrosa are the colour of the ripe oospores (golden-brown in C. fibrosa ssp. flaccida and black in the other two: C. fibrosa ssp. benthamii and C. fibrosa ssp. gymnopitys) and the number of stipulodes (as numerous as the branchlets in C. fibrosa ssp. benthamii, twice as numerous as the branchlets in C. fibrosa ssp. gymnopitys).
In the examined specimens of C. pelosiana, the stipulodes were variable in number, sometimes nearly equal and sometimes more or even twice as numerous as the branchlets ( Figure 9D,G and Figure 10A). Avetta reported that the number of stipulodes was equal to the number of the branchlets (10-12) [1] (pp. 232-233), while Langangen (despite having difficulty counting them) stated that there were 1-2 stipulodes per branchlet in the fragments of C. pelosiana that he observed in Oslo [6] (p. 250). It appears, therefore, the number of stipulodes in C. pelosiana is not constant, as has already observed by several authors in other species [7] (p. 154).
In contrast, the colour of the mature oospores in C. pelosiana was consistently found to be yellow-brown ( Figure 9A,E).
Only one stipulode per branchlet is mentioned in the description of the type material of C. fibrosa, and its oospores are described as being "consistently a light golden-brown" [15]. Furthermore, C. fibrosa is endemic to the island of Guam (Micronesia) [15].
This investigation of Chara fibrosa led to the separation of two previously merged species in the section Agardhia, C. fibrosa and C. wightii (A. Braun) Casanova [15]. Thus, the correct identity of C. pelosiana cannot be determined until the section Agardhia will be fully revised. Meanwhile, in this work, we used the valid name Chara pelosiana established by Avetta [1].

Conclusions
This study of herbarium materials of Chara pelosiana revealed unexpected original material. Only the C. pelosiana specimen described by Avetta [1] is mentioned in the literature. Although Ferrari's original collection is no longer kept in the Herbarium of Rome, two duplicates of the original set were discovered in the Modena and Turin Herbaria, each including a specimen of Chara pelosiana that can be considered "original material". Furthermore, two new additional localities were discovered in the Pavia and Turin Herbaria (rice fields of Campo Maggiore in the Province of Pavia and rice fields of Nonantola in the Province of Modena), providing new information about the Italian distribution area for this rare species. This paper gives an example of how, in addition to traditional morphological, taxonomic, and systematic research rules, historical herbarium collections can be used to assist ecology, biogeography, and conservation biology research.