Allozymic, Morphological, Phenological, Linguistic, Plant Use, and Nutritional Data of Benincasa hispida (Cucurbitaceae)

Allozymic, Morphological, Phenological, Linguistic, Plant Use, and Nutritional Data of Benincasa hispida (Cucurbitaceae)

Article  in  Economic Botany · March 2007

DOI: 10.1663/0013-0001(2007)61[44:AMPLPU]2.0.CO;2

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and Nutritional Data of Benincasa hispida (Cucurbitaceae)

Author(s): Kendrick L. Marr, Yong-Mei Xia, Nirmal K. Bhattarai Source: Economic Botany, 61(1):44-59. 2007.

Published By: The New York Botanical Garden

DOI: http://dx.doi.org/10.1663/0013-0001(2007)61[44:AMPLPU]2.0.CO;2 URL: http://www.bioone.org/doi/

full/10.1663/0013-0001%282007%2961%5B44%3AAMPLPU%5D2.0.CO%3B2

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Benincasa (Cucurbitaceae) is a monotypic genus with a single species, B. hispida (Thunb.)

Cogn. This annual vine, native to the Asian trop- ics (including India), is known almost solely as a domesticate and is grown primarily in the Old World tropics; to a lesser extent, it is also grown in the New World for its edible fruit (Walters and Deckers-Walters 1989). English names include

and Nutritional Data of Benincasa hispida (Cucurbitaceae)

¹

Kendrick L. Marr

^2,3,*

, Yong-Mei Xia

³

, and Nirmal K. Bhattarai

⁴

2Museum of Anthropology, University of Michigan, 4009 Museums Building 1109, Geddes Avenue, Ann Arbor, Michigan 48109-10792

3Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yun- nan, China 666303

4National Herbarium Plant Lab, G.P.O. Box 938, Godhavari, Lalitpur, Nepal

*Corresponding author: Current address: Department of Natural History, Royal British Columbia Mu- seum, 675 Belleville Street, Victoria, BC, Canada V8W 9W2; e-mail: kmarr@royalbcmuseum.bc.ca.

Allozymic, Morphological, Phenological, Linguistic, Plant Use, and Nutritional Data of Benincasa hispida(Cucurbitaceae).Benincasa hispida, from tropical Asia, is cultivated pri- marily for its edible fruit. To explore its domestication and ethnobotany, we made collections from two regions where it may have been domesticated: (1) Southern China/Southeast Asia, represented by southern Yunnan Province (China) and northern Laos; and (2) the In- dian subcontinent, represented by southeastern Nepal. Domestication has resulted in di- verse fruit sizes, shapes, and colors; seed sizes and shapes; and, in some accessions, the loss of tendrils. Modern cultivars have larger fruit, earlier flowers, and lower nutritional content than traditional cultivars. Allozyme diversity was low, divergence between these two regions was low, and these results do not clarify the place of domestication. The greater morpho- logical diversity of plants from Yunnan and Laos, as compared to Nepal, suggests domesti- cation in the former region. In all accessions, male flowers precede females. Benincasa hisp- idais prominent in some traditional stories and rituals.

Economic Botany, 61(1), 2007, pp. 44–59.

© 2007, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.

Key Words: Benincasa hispida, Cucurbitaceae, domestication, ethnobotany, morphology, phenology, nutritional content, tendrils, China, Nepal, Laos, wild.

1Received 31 August 2006; accepted 12 October 2006.

waxgourd, wintermelon, white gourd, ash gourd, white pumpkin, ash pumpkin, Chinese preserving melon, and hairy melon (when immature). Many of these names allude to the powdery white flakes of wax that coat the fruit surface of some culti- vars. The plants are generally monoecious; how- ever some cultivars bear hermaphrodite flowers (Roy and Saran 1990). The yellow flowers develop six to nine weeks after seed germination and the fruit matures two to three months later (Walters and Decker-Walters 1989). Hairs that cover the ovary and young fruit are lost as it matures. Fruit of modern cultivars are up to 2 meters (m) long and 45 kilograms (kg) (Rifai and Reyes 1993).

The mature and immature fruit (“hairy melon”) are edible. The flesh is white to pale green, with a rather bland flavor. In China and Southeast Asia, chunks of the mature fruit are commonly prepared in soup and, in India, chunks are cooked in curries (Rifai and Reyes 1993). In China, India, and Cuba, fruit chunks are “can- died” (Heiser 1979) and, in China, a canned bev- erage is made from the fruit. “Hairy melon” is sliced and eaten raw or cooked (Walters 1989).

In addition to the edible fruit, the flowers, young leaves, and seeds are edible; the fruit rind, seeds, roots and leaf juice have medicinal usages (Burkhill 1935; Yang and Walters 1992). In Poly- nesia, the dried exocarp of the probable wild type (see below) was (and evidently continues to be) used to store scented coconut oil (Whistler 1990).

The Sanskrit word for B.hispida, “kumbha-phala”

means “water pot fruit” (Decker-Walters 1998), which also suggests this use.

Benincasaappears in mythology, rituals, and id- ioms, perhaps due to the life-giving properties sometimes ascribed to “gourds” (Trankell 1995).

In a story of the Yao people (in China), a B.hisp- idafruit was the progenitor of all human beings as the offspring of the sun and the moon. In another Yao story, a woman and her brother were the sole survivors of a flood. Together, they gave birth to a B.hispida fruit, chunks of which became the an- cestors of all people (Marr and Xia 2001). “Pump- kins” of Asian creation stories probably refer to B.

hispida instead of Cucurbita sp. (Marr and Xia 2001). In some rituals of Nepal (Majupuria 1988) and India (Prabhakar 1995), the fruit may take the place of animal sacrifice.

The place of domestication of Benincasa hisp- idais unclear (Li 1970) in part because, until re- cently, the wild plants were thought to be extinct (Jeffrey 1980; Nazimuddin and Naqvi 1984; Rifai

and Reyes 1993). DeCandolle ([1886]1959) re- ported the occurrence of wild plants in Japan, Java (along the seashore), Australia, and the Society Is- lands and “that the cultivation of Benincasa in China dates from remotest antiquity.” According to Burkhill (1935), B.hispidawas “probably a na- tive of Malaysia” and occurred in the wild in Java.

It was definitely cultivated in China by 400–500 A.D. and possibly by 206 B.C. (Walters and Decker-Walters 1989). Walters and Walters (2000) demonstrated that B. hispida seeds re- mained viable in buoyant fruit kept in salt water for six months, confirming the possibility of dis- persal across great distances of ocean.

Recently, what appear to be wild B. hispida plants have been found. Whistler (1990) collected a plant from Tonga with round gray, waxy fruits measuring 5 to 12 centimeters (cm) in length, and seeds 7 to 10 millimeters (mm) long (with ridged margins). Because of its relatively small fruit, this presumably wild plant was named B.hispida var.

pruriens(Parkinson) Whistler (Whistler 1990). In 1996, probable wild plants were observed on Su- lawesi (Indonesia) at three locations below 50 m above sea level (Matthews 2003). These fruit, which were not bitter, measured 6 cm in diameter and had a waxy coating (seed measurements were not stated) (Matthews 2003).

There are few archaeobotanical remains of Benincasa. Seeds of B. hispida were found in Thailand in cultural levels dated to 9,980 to 9,530 years BP; however, they probably are mod- ern intrusives (Pyramarn 1989). In 1994, B.hisp- ida exocarp fragments and seeds measuring 7.5 mm to 8 mm by 4 mm to 4.5 mm (ridged margins) were found in a New Guinean agricul- tural site dated to 2,450 (± 200) years BP (Muke and Mandui 2003). It is unclear whether these were the wild type or the domesticate (Matthews 2003). In Japan, B. hispida seeds from archaeo- logical sites date from ca. 300 A.D. to the 1800s (Matthews 2003).

In this study our objectives were to: (1) use al- lozymic and morphological variation to gain in- sight into the place of domestication of B. hisp- ida; (2) document its genetic and phenological diversity; (3) analyze the nutritional content of different cultivars; and (4) document its utiliza- tion, cultural significance, and local names.

We collected samples from two regions: (1) tropical southwest Yunnan Province (China) and northern Laos to represent Southern China/SE Asia; and (2) tropical, southeastern Nepal to rep-

Table 1.Collection Location Numbers of Benincasa hispidaAccessions Collected From China (Yunnan Province), Nepal, Laos1. Collection Allozyme Location NumberVillagePrefecture/District*Elev. (m)Type#Ethnic GroupLocal NameAnalysis N/S China (Yunnan Province) C1ManMoXishuangbanna/JingHong700tAinitaoho4/8 C2GuoFaXishuangbanna/JingHong—tAini—6/12 C3DakaXishuangbanna/Mengla600tAini—13/26 [C4]HuidaXishuangbanna/JingHong1,100tAinitaoho— C5XiangDuoYaXishuangbanna/Mengla800tAini—4/8 C6NanLaXiaozhaiXishuangbanna/Mengla660tAinitohuo11/22 C7BaLoXishuangbanna/Mengla660tAini—4/9 C8JiuLongXinXishuangbanna/Mengla1,300tAinitohuo8/16 C9XinXiuHuaXishuangbanna/Mengla650tAinitohuo4/8 C10ManSaXishuangbanna/JingHong1,100tBulangtepil,enbi3/6 C11XinMinXishuangbanna/Mengla950tYaosop3/6 C12PaoZhuQingXishuangbanna/Mengla—tYaozhambu4/8 C13YiYuanShuiXishuangbanna/Mengla950tYaozhambu9/18 C14NalomXishuangbanna/Mengla930tMiaodaodu1/2 C15YaNuoXishuangbanna/JingHong1,130tJinuotoko6/12 C16BaKaXishuangbanna/JingHong650tJinuotuku tupulu4/8 C1747kmSimao/Lancang900tLahupemepexi6/13 C18CaiLongXinZhaiSimao/XiMeng800tWabilai1/2 C19XiaJiLiLincang/CangYuan1,150tWa—3/5 C20DaWanTianLincang/ZhengKang1,000tHandonggua6/12 C21GongWaiYiSheDeHong/JieLe1,450tJingpohongjiang1/2 C22GeDouDeHong/YingJiang800tJingPohongjiang1/2 C23BangJiaoDeHong/JieLe1,400tJingPohongjiang4/7 C24DoMenDeHong/JieLe1,000tJingPo—1/2 C25BaiShiTouDeHong/YingJiang1,200tJingPo—1/2 C26ShuangKueiDiNuJiang/LiuKu1,300tLisuapeme2/2 C27MangMaoLincang/Lincang1,300tHan/Dai—6/11 C28LuoLiangSimao/JingDong900tHan/ Yipeme1/1 C29GuoMuDehong/Luxi800tDaibapmoun4/8 C30HuiQingXishuangbanna/Mengla900tDai/Ainifakmuen6/12 C31NakaXishuangbanna/Mengla700tDaifakmuen5/9

C32MaliXishuangbanna/Mengla700tDaifaxmuen3/6 C33marketXishuangbanna/JingHong—mhandonggua1/2 C34##PI419170China—m-—1/2 C35marketXishuangbanna/JingHong—mdonggua—1/4 C36YuanJiang MktYuXi/YuanJiang—mdonggua—1/2 C37ManShi Mkt.DeHong/Luxi—tDaibakbuen1/2 [C38]XiaoJieDaMengLong—mhandonggua— [C39]Simao MktSimao/Simao—mhandonggua— C40+##PI384538——m——1/2 [C41]YingchangXishuangbanna/Mengla—tJinuotuku— [C42]SaiyouXishuangbanna/Jinghong900tAini— [C43]DaqingshuXishuangbanna/Mengla620tAini— [C44]LongbaXishuangbanna/Mengla∼800tYaozhambu— [C45]SousanjiaoXishuangbanna/Mengla1250tYaozhambu— [C46]HuiduXishuangbanna/Mengla850tYao/Dai/Handonggua— [C47]BayaxinXishuangbanna/Jinghong800tJinuotoko— [C48]ManzhangXishuangbanna/Mengla550tDaibakmuen— [C49]ManeXishuangbanna/Jinghong570tDaibopmuen— [C50]KongmudanDehong/Jiele1,400tJingpohongjiang— [C51]PingtianNujiang900tDaiBakbuen— [C52]XibenxiangLincang/Lincang1700tHandonggua— Laos L1BannasayOudomxai∼1,000tHmongdaodo3/6 L2LaksipOudomxai∼1,100tKhmu—3/6 L3HuikomOudomxai∼1,000tLaolongmakbuop4/8 L4BanpuonsaatOudomxai∼1,000tLaolongmakbuop4/8 L5NabaOudomxai∼900tLuamakduon9/18 L6BanmokokOudomxai∼1,000tHmongdaode9/18 L7BansamakisaiOudomxai∼1,000tLaotengbiergluk5/10 L8NametOudomxai∼1,000“w” ——2/4 L9MenghuenOudomxai∼1,000“w”Laolongmakfakkom1/2 Nepal N1NarayangadhNarayani/Chitwan200tNepalikhubindo3/5 N2KalikatarLumbini/Tanahu500tNepalikhubindo2/4 N3PathalaiyaNarayani/Bara200tNepalikhubindo1/2 N4BhataNarayani/Parsa200tNepalikhubindo7/10 N5BiratnagarKosi/Morang800tNepalikhubindo3/5 (continued)

resent the Indian subcontinent (logistical issues prevented the collection of samples from India).

In these regions, a great diversity of crops and tra- ditional cultivars are raised, e.g., 70 named rice cultivars, by one ethnic group (Long et al. 1995).

Methods and Materials

Germplasm and Data Collection

in the Field

We conducted unstructured interviews and collected seeds in many villages, each of which was given a collection location number (Table 1).

We made collections in southwest Yunnan from September 1997 to March 1998 and January to March 1999, in the Oudomxai Province of northern Laos in December 1997 and February 1999, and in Nepal from November 1998 and February 1999 (Fig. 1). Each seed collection was given a unique accession number. If a household offered seeds of different fruit, each was given a separate accession number.

Villagers usually described their plants as “old varieties” or “local varieties”; we refer to these as

“traditional” cultivars. We also purchased from markets or ordered seeds of commercial cultivars that we refer to as “modern” cultivars (Table 1).

We received seeds of the small-fruited Benin- casa from Tonga (that of Whistler 1990). Unfor- tunately, they did not germinate when we at- tempted to grow them.

Morphological Analyses

In the field, we recorded fruit length, width, and color whenever possible. We recorded seed Table 1.Continued Collection Allozyme Location NumberVillagePrefecture/District*Elev. (m)Type#Ethnic GroupLocal NameAnalysis N/S N6BudhabareMechi/Jhapa200tNepalikhubindo1/2 [N7]QuadiGandaki/Tanahu500tGurungkhubindo— [N8]BarahachhetraKosi/Sunsari∼300tNepalikhubindo— Thailand T1ChiangMai—————— 1All collection locations except those in “[ ]” were analyzed for allozyme diversity. For each collection location, “N” designates the number of accessions that were collected and “S” designates the number of individuals that were assayed for the allozyme analysis. * “Prefecture” is equivalent to a Laotian “Province” and a Nepali “Zone”. # t=traditional cultivar, m=modern cultivar, w=wild population ## Accession number of U.S. Dept. of Agriculture-Agriculture Research Section (USDA-ARS) +Seeds from United States, possibly originally from China

Fig. 1. Black-shaded areas indicate the general re- gions for which Benincasa hispidaseed collections were made for this study, in Nepal, China, and northern Laos.

length, width, mass, and margin type (ridged ver- sus smooth) of all accessions. To measure leaves and flowers, we grew plants from the seeds of a subset of the accessions that we had collected.

The accessions we chose represented the full range of geographical and morphological diver- sity as understood from fruit/seed characters.

Seeds were planted in a level, irrigated field at the Xishuangbanna Tropical Botanical Garden (XTBG) of the Chinese Academy of Sciences.

This institute is located at 21^o 41' N and 101^o 25' E at an elevation of 570 m, where the mean annual temperature is 21.4^oC. For each planted accession, we took various measurements of three to five leaves and flowers. Four ratios were calcu- lated to describe differences in leaf shape (Fig. 2).

For each accession, these measurements were av- eraged for subsequent analyses.

To summarize the morphological variation and to assist in visualizing possible regional differ- ences, we used Principal Components Analysis (PCA) of two sets of data with SYSTAT (1997).

The following characters were used in the first PCA of 233 accessions: seed length, width, weight, and margin type, and fruit length, width, and skin color. In the second PCA of 51 acces- sions (fewer than the first because fewer acces- sions had leaf data), we used all of the characters of the first PCA plus the four leaf ratio charac-

ters. For some collection locations, more than one accession was included.

Allozyme Analysis

We used cellulose-acetate gel electrophoresis following Marr, Xia, and Bhattarai (2004) to in- vestigate allozyme diversity (details available from the first author). Twenty-three loci of 16 enzymes were resolved: three for MDH; two each for MNR, PGM, PGI, AAT, and GDH; and one each for ALD, ADH, G6PDH, ACON, ME, 6PGD, LAP, SKE, IDH, and FDH. We used BIOSYS-1 (Swofford and Selander 1989) to pro- duce a phenogram based on Nei’s (1978) genetic identities (I).

Phenological Observations We planted (at XTBG) seeds of 23 traditional cultivars from China and Laos, and seven mod- ern cultivars from China on 17 March 1998. For the next few weeks, until the plants died due to flooding, we recorded the gender of the flower that appeared at each node.

Nutritional Analysis

The staff of the Yunnan Academy of Agricul- tural Sciences in Kunming (Yunnan) analyzed the fruit (flesh) nutritional content of seven tradi- tional cultivar accessions from China and Laos (raised at XTBG) and one modern cultivar each from Thailand (raised at XTBG) and China (pur- chased from a market in Kunming). The samples were oven dried and the moisture content, pro- tein, fiber, total sugars, S, Mg, Fe, Zn, P, K, and Ca measured. Total sugar was analyzed using the Fehling Method, protein by the Semimicro Kjel- dahl Method. Fiber was determined by first pro- cessing with dilute acid and alkali to eliminate saccharides and proteins and then with acetone to remove tannin, pigments, and remnant fats.

After the sample was heated to 300° C, the ash was weighed. Inorganic elements were analyzed by ICP-AES (Inductivity Coupled Plasma Atomic Emission Spectrum).

Results

Germplasm and Data Collection in the Field

We collected seeds of 293 accessions, 216 of which were from China, 42 from Laos, 34 from Nepal, and one from Thailand (Table 1; Appen- dix 1). In villages, we often removed the seeds di- Fig. 2. Outline of Benincasaleaf. Numbers indi-

cate dimensions that were measured. The following ra- tios are presented in Table 2: Ratio 1 =¹⁄₆; Ratio 2 =¹⁄₂^; Ratio 3 =³⁄₄; Ratio 4 =¹⁄₅^.

rectly from the fruit and the remainder of the fruit was fed to pigs or eaten by the family. Some- times we were given stored, dried seeds. Seeds of most accessions were stored at 4^oC in the seed bank of XTBG.

In China and Laos, traditional cultivars of B.

hispidawere common, planted in home gardens or in swidden fields with rice, corn, or cotton.

Some villagers stated that modern cultivars would not grow in their swidden fields. In one Dai vil- lage,B.hispidawas also planted in level rice fields after rice harvest. The fruit were common in mar- kets, often sold by the slice. In larger cities, these were modern cultivars, typically larger than the traditional ones. In smaller rural markets, both modern and traditional cultivars were sold.

Traditional cultivars of B. hispida were also common in Nepal and often were trained to grow onto rooftops. One farmer commented that it was not favored because it took up too much space and did not bear fruit as quickly as Cucurbitasp.

Fruit were globose or oblong and had yellow (rarely), green, or gray skin (Fig. 3). Of the 263 accessions whose fruit we observed, 195 were powdery gray, 59 were green, and nine were yel- low. Yellow and green fruit lacked powdery wax

flakes but were nevertheless waxy. In Nepal, all fruit were gray and oblong. In China and Laos, fruit of all three colors and both shapes occurred, often within a single village.

Seeds varied greatly among accessions (Fig. 4).

Nearly all seeds were glabrous, except for some modern cultivars and one putative wild plant (see below) that had pubescent (short, thick, straight hairs) seeds. Small fruit did not necessarily have the smallest seeds. Of the 293 accessions, seeds of 39 had smooth margins. Both seed margin types occurred in modern and traditional cultivars. All seeds from Nepal had ridged margins. Twenty- four of the 263 accessions whose fruit we ob- served had seeds with smooth margins. Of these 24, most were oblong, 22 were gray, one was yel- low, and one was green.

We could recognize ca. 16 cultivars based on fruit shape, length, width, and skin color. In most villages, especially in Laos and Yunnan, at least two cultivars were raised; sometimes as many as five were raised by a single family. Villagers de- scribed the fruit according to taste, size, shape, color, yield, phenology, cooking time, storage life, the thickness of the skin, and (rarely) seed margin types. In general, they preferred the taste of tradi- Fig. 3. Mature fruits of traditional cultivars of Benincasa hispidaillustrating part of the range of variation present in Oudomxai, Laos. The smallest fruit are from free-living plants. The ruler is 15 centimeters (cm) long.

tional cultivars. We were told that the fruit of tra- ditional cultivars could be stored for one to two years if the waxy surface was not rubbed off (oth- erwise the fruit would quickly spoil).

Several villagers in China and Laos spoke of a B.

hispidathat lacked tendrils (atypical for the Cucur- bitaceae) and that “climbed the ground.” We planted seeds of such plants. Their vines did not produce tendrils, or the tendrils were very short.

One farmer commented that in upland fields, vines without tendrils could not wrap around rice plants, making the rice easier to harvest.

We asked villagers if different cultivars were grown together and whether they flowered at the same time. The answer to both questions was al- ways affirmative. This suggested the potential for cross-pollination, therefore we asked if the culti- vars bred true. We were almost always told that they did breed true. When asked the same ques- tions regarding Cucumis sativusL., villagers stated that it did not breed true. This illustrates their

careful observations of cultivar diversity and sug- gests that their statements regarding B. hispida were accurate.

In China, most villagers had not seen wild B.

hispida; however, a ca. 60-year-old Bulang man told us that the Dai medicinal literature describes the use of a kind of B.hispidawith bitter fruit, a possible reference to the wild type. In Menghuen (Laos), four or five families planted, for medicinal preparations, a “wild” B.hispidathat they named

“makfakkom” (“kom” = bitter). They stated that such plants did not grow in the forest and the fruit was too bitter to eat. The fruit or the pedun- cle was eaten to treat nausea caused by eating bad food or for a woman who might be dizzy after childbirth. Does the peduncle symbolize the um- bilicus? They also used it to treat fever and as a

“cooling” substance to restore the body’s balance.

The only seeds of it we saw were 9 mm by 6 mm with ridged margins and covered by a thick pu- bescence (Fig. 4). Two fruit from Namet (Laos) were also supposedly wild and had a bitter taste.

These were 10 cm long by 11 cm to 12 cm wide, with green skin (Fig. 3) and 9 mm by 5 mm seeds (ridged margins).

We refer to the above plants as free-living (Table 1) because we were not certain that they were genuinely wild due to their large size (and absence of gray waxy coating) relative to the ap- parently genuine wild type from the Pacific (Whistler 1990) and Indonesia (Matthews 2003).

Usage

The primary usage was of the mature fruit for food. In China, chunks of the fruit are prepared as a soup, often with pork. Less commonly it is fried or eaten raw. Leaves were sometimes report- edly edible. In Nepal, the fruit are pickled, pre- pared in curries, or cooked, sliced, dried, and then mixed together with mango fruit and formed into a ball to fry later.

We were told several uses that relate to health.

Hmong place the fruit juice on the head to treat fever. Bulang believe that if they eat the fruit when they have malaria they will become sicker. Jingpo eat a gray-skinned cultivar if something tastes bad.

The Laoteng used the fruit together with the bark of a tree to prepare a beverage to help a woman keep a baby if she was not healthy. In Nepal, the fruit are “cooling” for people and bulls, and the immature fruit can be used as an abortifacient.

Perhaps because of the latter belief, pregnant cattle and buffalo are not allowed to eat it.

Fig. 4. Seeds of Benincasa hispida. Top three rows are from Yunnan, next two rows are from Laos, and the bottom row is from Nepal. All are traditional cultivars unless noted. Top row, left, first two seeds are modern cultivars. Third row from bottom, seed at far right is of putative wild plant from Laos (collection location L9, accession # L3443). The ruler is 15 centimeters (cm) long.

Names and Cultural Significance In China and Laos, modifications of the root word for B.hispidadescribed fruit skin color, flavor, and shape. Some closely resemble “pumpkin” (Cu- curbitasp.). In the Jinuo language, B.hispidais “tuku tupulu” and pumpkin is “tuku tungelu,” but both were often called “tuku” (“tuku”/“tu” = squashes/

melons; “ngelu” = red; “pulu” = gray/green). In Dai

“fak” or “mak” described squashes/melons, e.g., pumpkin, and “fakmuen” or “bakmuen” (“muen” = powder) described B.hispida. In the Lahu language,

“pemexi” described pumpkin and “pemepexi”

(“pe” = white) described B.hispida.

The Yao people have several stories or idioms that involve B.hispida. An unattractive woman is a “dong-gua” (Mandarin word for B. hispida fruit). An ill person who claims to be well is a

“dong-gua,” i.e., hard on the outside, soft on the inside. In one story, a man’s wife sent his younger brother to the field to bring back “dong-gua.” Re- turning with a bag of fruit, he became tired, so he took the fruit out of the bag, turned the bag in- side out, crawled into it, and slept. Some mon- keys came along and saw the bag that they took to be a large waxgourd (the outside was white be- cause of the powdery wax). They took it to their cave and placed it with other treasures. When the man awoke, he crawled from the bag and cried out, causing the monkeys to flee. He picked up the other treasures and took them home.

Some ritual significance is attached to the fruit.

Because white is associated with death, some Bu- lang and Aini people do not eat it when they are ill or when a person dies. When the Bulang build a new house, they cover a waxgourd and a pump- kin with a net. The colors of these fruits symbol- ize silver and gold. The net prevents the wealth from leaving. The Dai people, who practice Bud- dhism, make an offering of a squash, cucumber, watermelon, or waxgourd when they build a new home. In Nepal, poor people or vegetarians can use the fruit as a substitute for animal sacrifice.

Morphological Study

We planted seeds of 59 accessions, including three free-living ones from Namet (Laos) and made herbarium specimens of most (Appendix 1). Some plants died before flowering, but we were able to make measurements of their leaves.

Ten plants produced fruit and all resembled the fruit from which their seeds came.

Table 2 documents the range of morphological

variation. Leaves and flowers had lower sample sizes because measurements were taken only of accessions grown at XTBG. Modern cultivars were larger than traditional cultivars for most characters. Some traditional cultivars from China and Laos had smaller seeds than those of genuine wild or free-living plants (Table 2).

In the first PCA, the first axis accounted for 41% of the variation with seed weight, length, and width contributing the most. The second axis accounted for 21% of the variation with seed margin type, fruit length, and fruit width con- tributing the most. As a group, traditional culti- vars from China and Laos exhibited considerably greater diversity than did traditional cultivars from Nepal (Fig. 5). Traditional cultivars from Nepal did not cluster apart from those of China and Laos. Free-living accessions from Laos clus- tered together and with traditional cultivars from Laos and China. The single modern accession was distinct from the others.

In the second PCA, the first axis accounted for 36% of the variation with seed length, weight, and width contributing the most. The second axis accounted for 23% of the variation with leaf ratio 3, leaf ratio 1, and leaf ratio 2 contributing the most. The third axis accounted for 17% of Fig. 5. Scatterplot of Benincasa hispidaaccessions from PCA of seed and fruit characters (see text). Each symbol represents a single accession, labeled by its col- lection type: C, L, N represent traditional cultivars from China, Laos, and Nepal, respectively; F represents free-living accessions from Laos, M represents a mod- ern cultivar.

Table 2.Leaf, Fruit, Seed and Flower Measurements of Benicasa hispidaCollected from China (Yunnan Province), Laos, and Nepal.1 Accession types Traditional* Wild Modern Plant partChinaLaosNepalFree-living*(literature**)Modern*(literature***) Leaf Length (cm)(34)10±2(13)11±1(5)13±1(3)6±1—(4)13±2— 6–148–1312–145–67–1611–1610–25 Width (cm)(34)14±1(13)14±1(5)19±1(3)8±1—(4)18±2— 8–2012–1816–208–17–1616–2210–21 Ratio 1(35)2.0±0.4(13)2.0±0.3(5)1.5±0.1(3)2.6±0.6—(4)1.8±0.3— 1.3–3.11.5–2.61.4–1.51.9–3.4—1.4–2.2— Ratio 2(34)1.5±0.1(13)1.5±0.1(5)1.5±0.3(3)1.5±0.1—(4)1.4±0.1— 1.2–1.91.4–1.61.3–2.01.5–1.6—1.3–1.5— Ratio 3(34)2.5±0.8(13)2.6±0.7(5)1.7±0.2(3)2.5±3.1—(4)2.3±0.6— 1.2–4.41.3–4.11.7–1.91.9–3.8—1.5–2.9— Ratio 4(34)1.1±0.2(13)1.1±0.3(5)1.2±0.2(3)1.4–0.1—(4)1.9±0.6— 0.8–1.70.6–1.61.0–1.51.3–1.6—1.4–2.5— Male Flowers Petal length (mm)(23)37±7(7)32±5(6)41±5(1)26—(2)35±14— 25–5123–3936–51—30–5025–4545–65 Sepal length (mm)(23)12±6(7)7±1(6)15±3(1)6—(2)13±8— 5–215–911–18—10–187–19 6–20**** Sepal width (mm)(23)8±2(7)8±1(6)9±2(1)5—(2)7±5— 3–117–97–13——3–10— Androecium length (mm)(23)9±2(7)9±1(6)11±1(1)5—(2)8±2— 6–137–1110–11——6–10— Pedicel length (mm)(23)68±24(7)54±9(6)111±15(1)46—(2)66±9— 35–12040–7186–131—10–5060–7250–150 Female flowers Ovary length (cm)(9)3.1±1.1(7)2.1±0.6(5)2.8±0.1(1)1.9—(1)2.6— 1.6–5.11.4–3.22.7–3.1———— Ovary width (cm)(9)1.4±0.2(7)1.3±2.3(5)1.6±0.2(1)1.6—(1)1.2— 1.2–1.61.0–1.71.4–1.8———— Petal length (mm)(9)40±8(7)39±9(5)48±6(1)35—(1)50— 30–5226–5341–55———40–60 Sepal length (mm)(8)7±1(7)5±1(5)9±2(1)5—(1)13— 5–94–67–12———— (continued)

Table 2.Continued Accession types Traditional* Wild Modern Plant partChinaLaosNepalFree-living*(literature**)Modern*(literature***) Sepal width (mm)(8)6±1(7)6±2(5)9±4(1)8—(1)8— 4–85–105–15———— Style length (mm)(8)13±4(7)11±2(5)15±1(1)13—(1)19— 8–188–1512–16———— Pedicel length (mm)(8)39±9(7)24±9(5)65±19(1)72—(1)16— 20–5111–3836–83———10–38 Fruit Length (cm)(184)36±10(34)32±9(31)32±8(3)11±1—(4)80— 16–8020–4525–5010–12——20–200 Width (cm)(184)17±6(34)20±6(31)20±3(3)11±1—(1)50— 8–5010–3012–2511–135–12—10–25 Seed mass (mg)(223)38±13(43)33±13(31)50±9(4)22±6—(10)48±12— 6–6710–5329–6714–29—26–6370 Seed length (mm)(216)9.5±2.0(42)10±2(37)11±0.8(4)9±1.0—(11)12±1.0— 4–146–1310–147–97–1011–1310–15 Seed width (mm)(216)5±1(42)5±1(37)6±1(4)5±1—(11)6.5±0.5— 3–94–75–93–64–4.56–75–8 Seed margin(226)1.1±0.3(43)1.2±0.4(37)1±0(5)1.2±0.4—(10)1.3±.5— (1=ridged; 2=smooth)1–21–21(1–2)—1–21–2 Shape(226)1.1±0.3(44)1.2±0.4(4)1(3)2—(10)1±.2— (1=oblong; 2=globose)1–21–2——1–21–21–2 Skin color (1=gray,(183)1.2±0.5(34)1.8±0.8(37)1(3)21(10)1±.2— 2=green, 3=yellow)1–31–3———1–21–2 1Values in ( ) are the sample size, beside the mean and standard deviation, range is beneath. See figure 2 for an explanation of the leaf measurements and ratio. * From current study ** From Whistler (1990), Matthews (2003), Walters and Walters (2000) *** From Walters and Decker-Walters (1989), Rifai and Reyes (1993) **** values are for both staminate and pistillate flowers

the variation with leaf ratio 4 and seed margin type contributing the most. As a group, acces- sions from Laos and China were more diverse than were those from Nepal (Fig. 6). Traditional cultivars from Nepal clustered together. Free- living accessions nested within the traditional cultivars from Laos and China.

Allozyme Results

We assayed 385 individuals (two for most ac- cessions) of 197 accessions from 52 collection lo- cations (Table 1). Isozymes were labeled sequen- tially with “1” as the most anodal locus and allozymes were labeled sequentially beginning with “a” as the most anodal.

Two loci, Mdh-3 and Pgm-1, were polymor- phic (two alleles each). Eleven of the 12 individu- als of modern accessions possessed Mdh-3a, a single plant possessed Mdh-3b, while all but three traditional accessions (from collection locations C6 and C7), possessed Mdh-3b. At Pgm-1 no in- dividuals were heterozygous. This was surprising Fig. 6. Scatterplot of Benincasa hispidaaccessions

from PCA of seed, fruit, and leaf ratio characters (see text). Each symbol is of a single accession, labeled by its collection location (Table 1). (L8 is a free-living ac- cession, C35 and C36 are modern, all others are tradi- tional).

Fig. 7. Phenogram based on Nei’s Genetic Identities for allozyme analysis of Benincasa hispida. Numbers rep- resent collection locations (see Table 1). Cophenetic correlation coefficient = 0.868.

since heterozygotes were expected because both alleles were present in several collection locations from China (18 of 32), Laos (five of seven), and Nepal (one of nine).

The extent of allozyme divergence among col- lection locations was very low (Fig. 7). Free-living accessions were not distinct from traditional, and modern cultivars clustered apart from both. The primary division in the phenogram is due to the dominant presence of Mdh-3a in modern acces- sions and the dominance of Mdh-3bin the tradi- tional and free-living accessions. Divergence among the branches of the traditional and free- living accessions is due to varying proportions of Pgm-1aversus Pgm-1b. These latter divisions ap- pear to not correspond with regional variation or even within regions.

Phenology Study

The first node to produce a flower varied from node 11–53 (Fig. 8). Most modern accessions flowered earlier than traditional ones and male flowers preceded female.

Nutritional Analysis

Nutrient concentrations varied among accessions (Table 3), but were generally higher than published values (Wills et al. 1984). Traditional cultivars gen- erally had higher concentrations of most nutrients.

Discussion

The greater morphological diversity of B. hisp- idaaccessions from southern Yunnan and north-

ern Laos suggests that this region was closer to the place of domestication than was southeast Nepal. The assumption here is that the area of greatest extant diversity is closest to the center of origin. Single villages and even single families in Laos and Yunnan raise a greater diversity of culti- vars than in all of southeast Nepal. This diversity was evident in the field (Fig. 3 and 4; Table 2) and in the PCAs (Fig. 5 and 6). Although com- paratively fewer samples were collected and ana- lyzed from Nepal, these were representative of the area that we visited. Additional support for an origin in the region of Yunnan and Laos is the presence of cultivars and free-living plants with small fruit, which are presumably the least evolved from the wild type.

Because the results from Nepal may not be representative of the entire Indian subcontinent, analysis of samples collected from India would be very useful. However, our observation that Nepal has only cultivars with gray skin may extend to the entire Indian subcontinent because Indian, Pakistani, and Nepali names for B. hispidaallude to a white waxy coating of the fruit and do not mention other colors (Hooker 1973; Majupuria 1988; Nazimmudin and Naqvi 1984).

The low level of allozyme diversity in B. hisp- ida, is similar to other Asian domesticated Cu- curbitaceae (Marr, Bhattarai, and Xia 2005;

Marr, Xia, and Bhattarai 2004; Marr, Xia, and Bhattarai 2005) and crop plants in general (Doebley 1989). In future studies, if it is found that free-living or wild populations possess more Fig. 8. Node at which first flower appeared for accessions of Benincasa hispidafrom China and Laos. Bars represent a single accession labeled according to its collection location (Table 1).

alleles than the domesticate, the absence of al- lozyme divergence among regions that was found in this study would suggest that domesti- cation had occurred in a single region. If the al- leles of populations from one region had been a subset of those of a second region, we could have proposed the latter region as the place of domestication, but only the Pgm-1 locus (and rarely Mdh-3) was polymorphic in the tradi- tional cultivars and both alleles were found in both regions.

The lack of heterozygotes at Pgm-1 in loca- tions where both alleles were present suggests the possibility of a barrier to gene flow among plants.

This could explain why traditional cultivars ap- pear to “breed true.” Seed formation via apomixis is also a possibility, as asexual reproduction does occur in the Cucurbitaceae (Singh 1990). Al- though Walters and Decker-Walters (1989) re- port that “various cultivars . . . interbreed freely,”

further study of B.hispida fertilization and seed formation would be useful.

Phenological variation among accessions of B.

hispida parallels two other domesticated Asian Cucurbitaceae (Marr, Bhattarai, and Xia 2005;

Marr, Xia, Bhattarai 2005). Male flowers pre- ceded female in these three species and is com- mon in many Cucurbitaceae (Condon and Gilbert 1990). The relatively early flowering of free-living B.hispidafrom Laos (L8 in Figure 8)

further suggests that it is not genuinely wild since this trait is likely a result of human selection.

The generally superior nutritional content of B.hispida traditional cultivars illustrates the im- portance of their conservation. As China devel- ops economically, genetic erosion of these and many other crop species is likely. This is less of a problem in Nepal and Laos, due to their greater reliance on subsistence agriculture.

Seed characters could potentially resolve the wild versus domesticate status of various B. hispida accessions because seeds of domesticated species are usually larger than their wild progenitor. How- ever, for B. hispida, the situation is confusing be- cause some domesticates had smaller seeds than both the wild plants from Tonga (Table 2) and the free-living plants from Laos. Seed size does not re- solve the status of the Indonesian plants from an archaeological site (Matthews 2003) because their size is within the range of both the Tongan plant and the domesticate (Table 2).

The recent discoveries of probable wild plants offer the possibility of understanding the place and process of domestication of B. hispidamore precisely than was previously possible. Further re- search on this question using more sensitive DNA markers would be useful and should in- clude tissue of the probable wild plants from Asia and the Pacific as well as traditional cultivars from India and other areas of Southeast Asia.

Table 3. Nutritional Content of Benincasa hispidaFruit.¹

Collection Water Protein Fiber Sugar P K Ca Mg Fe Zn Location (%) (%) (%) (%) (%) (%) (%) (%) (mg/kg) (mg/kg)

Traditional Cultivars

C30 94 18.6 13.3 25.0 0.67 3.86 0.14 0.13 53 26

C22 92 10.9 11.7 25.6 0.28 3.59 0.15 0.12 65 30

C29 94 9.9 11.2 26.7 0.32 3.65 0.20 0.17 82 34

C8 92 16.0 15.2 12.1 0.30 3.65 0.21 0.18 67 29

C29 95 11.8 11.2 20.6 0.25 3.67 0.22 0.16 73 15

L6 92 24.6 17.3 12.5 0.49 5.23 0.21 0.15 104 89

N5 — 15.7 8.5 — 0.49 6.43 0.45 0.19 105 44

Modern Cultivars

T1 95 12.3 14.5 31.5 0.27 3.69 0.20 0.13 52 22

Kunming 97 6.6 15.49 27.9 0.18 3.81 0.23 0.10 83 17

market

*hairy melon 93 11.3 — 27.4 — 4.0 0.19 0.24 48 32

*wax gourd 97 9.3 — 31 — 2.4 0.08 0.12 94 63

1Dry weight of edible portion of a single accession from indicated collection location; see Table 1 For collection locations.

*Wills, R. B. H., A. W. K. Wong, F. M. Scriven, and H. Greenfield. 1984. Nutrient composition of Chinese vegetables. Jour- nal of Agricultural and Food Chemistry 32: 413–416.